Method of treating multiple sclerosis with anti-K6 antibody

ABSTRACT

Methods and kits for treating inflammatory conditions are described that include modulating kallikrein 6 protease activity.

This application is a National Stage application under 35 U.S.C. §371that claims the benefit of PCT/US03/16106, filed 21 May 2003, whichclaims the benefit of U.S. Provisional Application Ser. No. 60/382,471,filed 21 May 2002.

TECHNICAL FIELD

The invention relates to methods and materials for treating inflammatoryconditions. More specifically, the invention relates to using modulatorsof kallikrein 6 protease activity to treat inflammatory conditions suchas multiple sclerosis.

BACKGROUND

Multiple Sclerosis (MS) is a demyelinating disorder that affects over350,000 persons in the United States today, with 8,000 new casesreported each year. MS is the most common chronic inflammatory diseaseinvolving the nervous system. The majority of people with MS arediagnosed between the ages of 20 and 50, although in rare cases,symptoms may appear in childhood or after age 50. MS is twice as commonin women than in men and more frequently diagnosed in Caucasians thanother racial groups. The cause of MS is unknown, although there isconsiderable evidence that it is an autoimmune disease. Although thedisease does not result in early death or impairment of cognitivefunctions, it can cripple the patient by disturbing visual acuity;stimulating double vision; disturbing motor functions affecting walkingand use of the hands; producing bowel and bladder incontinence;spasticity; and sensory deficits, such as touch, pain, and temperaturesensitivity.

Patients typically are diagnosed based on a combination of patienthistory and neurologic examination, including magnetic resonance imaging(MRI) of the brain and spinal cord, electrodiagnostic procedures (e.g.,evoked potential tests such as visual evoked potentials, brain stemauditory evoked potentials, or somatosensory evoked potentials), andlumbar puncture to look for evidence of immunoglobulin synthesis in thecerebrospinal fluid.

Currently, there is no cure available for MS, so treatment typicallyinvolves management of symptoms and treatment of the frequency andseverity of relapses. Therapeutics that have been approved since 1993include interferon-β for use in ambulatory patients withrelapsing-remitting MS (Paty et al., Neurology 43:662-667, 1993) (e.g.,Betaseron® (recombinant interferon β-1β) and Avonex® (recombinantinterferon β1α); glatiramer acetate (Copaxone®) for relapsing-remittingMS; and Novantrone® for secondary progressive and relapsing-remittingdisease.

SUMMARY

The invention is based on the discovery that modulators of kallikrein 6(K6) can alter pathogenesis of inflammatory cell mediated diseases bothwithin the central nervous system (CNS) and in the periphery, and as aresult, can aid in the treatment and prevention of inflammatoryconditions such as MS, rheumatoid arthritis, lupus, and asthma. Asdescribed herein, an antibody having specific binding affinity for K6reduced the degree of demyelination and reduced behavioral deficits inanimal models of multiple sclerosis.

The invention features a method for treating an inflammatory conditionin a mammal. The method includes administering to the mammal an amountof a K6 modulator effective to treat the inflammatory condition, and canfurther include monitoring the inflammatory condition in the mammal. Theinflammatory condition can be selected from the group consisting ofmultiple sclerosis, rheumatoid arthritis, lupus, and asthma. The methodis particularly useful for multiple sclerosis. The K6 modulator can bean antibody having specific binding affinity for K6. The antibody can bepolyclonal or monoclonal, and can inhibit the enzyme activity of K6. TheK6 modulator can be an antisense nucleic acid that inhibits theexpression of K6. In some embodiments, the K6 modulator is a peptidenucleic acid that inhibits the expression of K6. The K6 modulator alsocan be a serine protease inhibitor.

In another aspect, the invention features an antibody that specificallybinds to human K6 and inhibits the enzymatic activity of K6 and kitscontaining such an antibody. The antibody can be polyclonal ormonoclonal. A kit further can include a label or package insertindicating that the antibody is useful for treating an inflammatorycondition.

The invention also features a method for screening a subject for aninflammatory condition. The method includes detecting the level of K6protein or RNA present in a biological sample from the subject; andcomparing the level of K6 protein or RNA in the sample to thecorresponding level in a control population, wherein an increase in thelevel of K6 protein or RNA in the subject relative to that of thecontrol population is indicative of the inflammatory condition in thesubject.

A method for monitoring therapy for an inflammatory condition also isfeatured. The method includes detecting the level of K6 protein or RNApresent in a biological sample from a subject undergoing treatment forthe inflammatory condition; and comparing the level of K6 protein or RNAin the sample to a baseline level of K6 present in the subject, whereina decrease in the level of K6 protein or RNA in the subject relative tothat of the control population is indicative of a positive response tothe therapy in the subject. The inflammatory condition can be selectedfrom the group consisting of multiple sclerosis, rheumatoid arthritis,lupus, and asthma. The biological sample can be selected from the groupconsisting of serum and cerebrospinal fluid.

The level of K6 protein can be detected immunologically. For example,the level of K6 protein can be detected using a monoclonal antibody. Thelevel of K6 also can be detected using a capture antibody and adetection antibody, wherein the detection antibody includes a label(e.g., a fluorophore such as fluorescein, fluorescein isothiocyanate(FITC), phycoerythrin (PE), allophycocyanin (APC), or peridininchlorophyll protein (PerCP); biotin; an enzyme; or a radioisotope. Thecapture antibody can be attached to a solid substrate (e.g., a bead or amicrotiter plate). The capture antibody can be a polyclonal antibody.

In another aspect, the invention features an antisense oligonucleotidethat inhibits the expression of K6, wherein the oligonucleotide is atleast 8 nucleotides in length. The oligonucleotide can be at least 15nucleotides in length.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

Other features and advantages of the invention will be apparent from thefollowing detailed description, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1 contains dark-field photomicrographs showing the autoradiographiclocalization of K6 mRNA in a transverse section through the spinal cordof a control rat, and in parallel sections of paired experimental ratsat 3 and 7 days after intraperitoneal injection of KA. DF, LF, and VF:Dorsal, Lateral and Ventral funiculi of the white matter.

FIG. 2 contains photomicrographs of normal white (nWM and gray matter(GM) in the spinal cord of control animals (left panel) and inassociation with inflammatory cells at sites of active demyelination 180days following TMEV infection (right panel). Oligodendrocytes are markedby arrows; bar=200 μm.

FIG. 3A is a photomicrograph of a well-demarcated chronic active MSlesion, (luxol fast blue/periodic acid Schiff's (LFB/PAS) myelin stain).FIG. 3B is a higher magnification view of FIG. 3A. FIG. 3C is a parallelsection to 3A, stained for K6-IR, which is upregulated withininflammatory cells at the border between the plaque (PL) and periplaquewhite matter (PPWM), on the side of the lesion (arrow). FIG. 3D is ahigher magnification view of FIG. 3C. bar=200 μm.

FIG. 4 is a graph of the amount of K6 detected in sera from uninfectedmice (control, solid circle), or in mice at 90 and 180 days post-TMEVinfection (open circles and solid triangles, respectively).

FIGS. 5A-5B are photographs of 7.5% SDS-PAGE (reducing) gels stainedwith Coomassie blue that depict degradation collagen type I (5A),fibronectin (5B), and laminin (5C). In FIG. 5A, lanes 1-6 are molecularmass standards kDa), gelatin+K6 0 min; 5 min; 30 min; 60 min; gelatinalone 60 min incubation, respectively. In FIG. 5B, lanes 1-5 aremolecular mass standards; fibronectin+K6 0 hr; 1 hr; 24 hr; fibronectincontrol 24 hr, respectively. In FIG. 5C, lanes 1-5 are laminin+K6 0 hr;1 hr; 16 hr; laminin control 16 hr, molecular mass standards,respectively.

FIGS. 6A and 6B are photographs of 16.5% Tricine SDS PAGE gels showingdegradation of rat myelin basic protein by K6 (11A) and rat αMOGexternal fragment (residues 1-125) by K6. In FIG. 6A, lanes 1-9 aremolecular mass markers; no K6; 0 min; 1 min; 2 min; 5 min; 10 min; 30min; 60 min incubation with K6, respectively. In FIG. 6B, lanes 1-6 aremolecular mass markers; 0 min; 1 hr; 4 hr; 12 hrs; and molecular massmarkers, respectively.

FIG. 7 is a graph of the number of migrated cells as a function oftreatment. *P<0.05, SNK.

FIG. 8 is a Western blot of K6 (arrow at 25 kDa) in proliferating OLGs(Pro), and after 48 hr, 72 hr, and 7 days in culture.

FIG. 9 is a Western blot showing the endogenous inhibitor of K6 isunique in CNS relative to non-CNS tissues. Mature K6 is marked by anarrow. Lanes 1-4 are 10 ng r-K6; 2.2 mg/ml spinal cord; 6.4 mg/ml brain;9.6 mg/ml kidney, respectively. The higher molecular weight complexesmay represent the formation of K6/inhibitor complexes, with masses ofapproximately 42 and 91 kDa. The larger 91 kDa complex is foundprimarily in the kidney (double asterisks), whereas the 42 kDa complexis found predominantly in the brain and spinal cord (single asterisk).

FIGS. 10A-10C are graphs of the number of differentiated oligodendrogliaafter exposure to different amounts of recombinant K6 in vitro. *p<0.05,SNK.

FIG. 11 is a graph depicting the K6 antibody response.

FIG. 12 is a graph depicting high K6-antibody titers, decreased spinalcord demyelination and inflammation in the TMEV model of MS.Quantification of the percent spinal cord quadrants with demyelination,or inflammation in animals 40 days post-TMEV infection, which had beenpreviously immunized with K6 or OVA in CFA, with CFA alone, or withoutany prior immunization (none). Immunization of mice with K6 (5 weeksprior to TMEV infection), significantly decreased the number of spinalcord quadrants associated with demyelination or inflammation, relativeto all immunization control groups (ANOVA, P<0.002, *SNK, P<0.05).

FIG. 13 is a graph indicating that K6-immunization results insignificantly less white matter pathology following TMEV infection.Quantification of the area of white matter pathology along the length ofthe spinal cord 40 days post-TMEV infection in K6-immunized micerelative to control mice immunized with OVA, CFA alone, or without anyprior immunization (none). K6-immunization was associated with a greaterthan 3-fold reduction in the percent of the spinal cord white matterassociated with pathology (ANOVA, P<0.002, *SNK, P<0.05).

FIGS. 14A and B are histograms quantitating the brain pathology incontrol animals immunized with CFA alone (A) or with K6 (B). Each symbolrepresents an individual mouse graded on a scale of 0 to 4. Cb,cerebellum; BS, brain stem; Str, striatum; Ctx, cortex; Hippo,hippocampus; CCall, corpus callosum; Mng, Meningeal Inflammation.

FIG. 15 is a graph depicting DTH responses to the disease-inducing TMEV.Data shown represent the mean 48 hr change in ear thickness±SEM, inresponse to challenge with 2.5 μg of UV-irradiated, purified TMEV. DTHresponses were significantly reduced in the K6-immunized mice at the 48hr time point (*P<0.05, unpaired Students t-test), compared to miceimmunized with CFA-containing PBS alone.

FIGS. 16A and 16B are graphs indicating that K6-immunization at the timeof EAE induction delays the onset and attenuates the severity ofPLP139-151 EAE. Mice were immunized with PBS or K6 at the time ofpriming with 100 μg of PLP139-151 in CFA. (A) Data represent meanclinical score of each group plotted against time. The clinical scoresof mice immunized with K6 were significantly below those of control PBSimmunized mice from D9 throughout the remainder of the disease courseexamined; * P<0.05, Mann-Whitney U test. (B) K6-immunization at the timeof priming was also associated with a significant increase in survival(10/28 compared to 22/28, P=0.003 χ2 using Fisher's exact test).

FIGS. 17A and 17B are graphs indicating that pre-immunization with K6delays the onset and development of clinical and histological disease inPLP139-151 primed mice. (A) The mean day of onset and the time to peakdisease were both delayed by 3 to 4 days in the K6 immunized mice,compared to non-immunized, and PBS-immunized controls (P<0.02). K6immunized mice also exhibited significantly reduced mean daily clinicalscores up to day 15 post-PLP priming (*P<0.001 Mann-Whitney U test). (B)Detailed histological evaluation of the spinal cord of mice in eachgroup at the 21 day time point, indicated that K6-preimmunizationreduced both of the extent of parenchymal pathology and meningealinflammation, compared to each of the control groups examined (*MannWhitney U, P<0.05).

FIGS. 18A-D are graphs depicting that pre-immunization with K6attenuates clinical disease, the development of CNS pathology, and thedevelopment of Th1 responses in vivo and in vitro when examined duringthe acute phase of the disease. (A) The severity of clinical disease wasreduced in mice pre-immunized with K6 compared to control groupsimmunized with PBS alone, or receiving no prior immunization (*MannWhitney U, P<0.05), (n=14 per group). (B) The number of quandrants ofthe spinal cord associated with pathology or meningeal inflammation alsowere significantly reduced in K6-pre-immunized mice relative to controlswhen examined at the 12 day time point (unpaired Student's t-test,P≦0.05). (C) Splenic lymphocytes were harvested from all mice examinedin (A), and viable cells (5×10⁵/well) were cultured with indicatedconcentrations of PLP139-151 for 4 days. Cultures were pulsed with^(3H)TdR 18 hrs prior to harvest. Data shown represent the combined dataof two separate experiments. Splenocytes harvested from K6-immunizedmice exhibited significantly less proliferation in response to thepriming antigen, compared to their immunization controls (*P≦0.005,unpaired Students t-test). (D) DTH responses to the initiatingPLP139-151 peptide were evaluated in all mice at day 9 after priming.Data shown represent the mean 24-hr and 48 hr change in earthickness±SEM, in response to challenge with 10 μg of PLP139-151peptide. DTH responses were significantly reduced in the K6-immunizedmice at the 48 hr time point (*P<0.05, unpaired Students t-test).

FIG. 19 contains graphs depicting a decrease in antigen specific Th1cytokine production in response to K6 immunization. Splenocytes werecultured from the animals described in FIGS. 18A to 18D on day 12 postEAE-induction and stimulated with the priming antigen, PLP-139-151, fora period of 72 hr. Cell culture supernatants were harvested and analyzedfor IFN-γ and IL-2 secretion by capture ELISA techniques. IFN-γproduction, a Th1 cytokine, was significantly reduced in K6-immunizedmice (*unpaired Student's t-test, P<0.05). By contrast, IL-2, a Th0cytokine, was comparable between the different experimental groups.

FIG. 20A is a photograph of a 20% Tricine SDS-PAGE (run under reducingconditions) gel showing hydrolysis of rat MBP in the presence or absenceof IgG isolated from K6-immunized mice or controls. Lanes: 1, molecularmass markers; 2 anti-K6 IgG+K6+MBP; 3 anti-CFA IgG+K6+MBP; 4 K6+MBP; 5anti-K6 IgG+MBP; 6 anti-CFA IgG+MBP; 7 MBP alone.

FIG. 20B is a graph depicting the rate of AcATRpNA-substrate hydrolysisover time by K6 in the presence of IgG isolated from K6 immunized miceor controls.

FIG. 21 is a graph depicting ability of monoclonal antibodies specificfor K6 to block K6 enzymatic activity in vitro.

FIG. 22 is a graph depicting that immuoglobulin isolated fromK6-immunized mice inhibits the migration of activated splenocytes invitro. Compared with normal mouse IgG (control) and to the addition ofno antibody, the addition of K6-IgG inhibited migration by 25%(*unpaired Student's t-test).

FIGS. 23A and 23B are histograms showing intracellular staining of K6determined by flow cytometry. The filled areas show constitutiveintracellular expression of K6 by CD4+ (A) and CD8+ (B) splenocytescultured in PBS.

FIG. 24 (left panel) and 24 (right panel) are graphs depicting thatsplenocyte activation resulted in an increase in K6-production andsecretion. Splenocytes were grown in media containing PBS, as a control(left panel) or in media containing 10 μg/ml Con A, 5 mg/ml LPS (rightpanel). Alternatively, flasks were pre-coated with 10 μg/ml CD3 antibody(Ab) for CD3 receptor cross-linking. Specific activation of T cells (ConA and CD3 Ab), or non-specific activation of all splenocytes, produced asignificant increase in K6 production, and secretion into the media,compared to those cells grown in PBS alone.

FIG. 25 is the nucleotide sequence (SEQ ID NO:2) encoding human K6.

DETAILED DESCRIPTION

In general, the invention provides methods for treating inflammatoryconditions in mammals using modulators of K6 as well as methods ofdetecting the presence of an inflammatory condition and monitoringinflammatory disease state by detecting the level of K6 protein or aribonucleic acid encoding K6 in biological samples from the mammals. Theterm “K6” as used herein refers to mammalian kallikrein 6 (e.g., frommice, rat, and humans). It should be noted that human K6 also isreferred to as protease M, neurosin, zyme, and myelencephalon-specificprotease (MSP). In the mouse, K6 also is referred to as brain and skinserine protease (BSSP) or brain serine protease (BSP). The nucleic acidsequence encoding human K6 can be found in GenBank under Accession Nos.AF013988, AF149289, and D78203. K6 is expressed in the CNS and withinthe CNS, is most abundant in the hippocampus, substantia nigra, basalganglia, and spinal cord. K6 exhibits a limited distribution innon-neural tissues. Within normal white matter, K6 expression is almostexclusively associated with oligodendroglia. K6 levels are up-regulatedin both neural and glial elements following injurious events, such asglutamate-receptor mediated excitotoxic injury.

Without being bound by a particular mechanism, K6 is localized withinboth macrophages and T cell subsets at sites of CNS inflammation anddemyelination and can degrade myelin-specific and extracellular matrixproteins, and when present in excess, negatively effect oligodendrocyteprocess outgrowth and integrity. K6 may facilitate transendothelialmigration of inflammatory cells into, and within, the CNS.

Methods of Treating Inflammatory Conditions

The term “inflammatory condition” as used herein refers to anyinflammatory cell mediated disease within the CNS or within theperiphery, including infectious (bacterial or viral) and autoimmunediseases. Non-limiting examples of inflammatory conditions affecting thenervous system include MS; all types of encephalitis and meningitis;acute disseminated encephalomyelitis; acute transverse myelitis;neuromyelitis optica; focal demyelinating syndromes (e.g., Balo'sconcentric sclerosis and Marburg variant of MS); progressive multifocalleukoencephalopathy; subacute sclerosing panencephalitis; acutehaemorrhagic leucoencephalitis (Hurst's disease); human T-lymphotropicvirus type-1-associated myelopathy/tropical spactic paraparesis; Devic'sdisease; human immunodeficiency virus encephalopathy; humanimmunodeficiency virus vacuolar myelopathy; peipheral neuropathies;Guillanin-Barre Syndrome and other immune mediated neuropathies; andmyasthenia gravis. Non-limiting examples of non-nervous systeminflammatory conditions include rheumatoid arthritis; osteoarthritis;infectious arthritis; psoriatic arthritis; polychondritis; periarticulardisorders; colitis; pancreatitis; system lupus erythematosus;conjunctivitis; diabetes type II; dermatitis; asthma; systemic sclerosis(scleroderma); Sjogren's syndrome; Behcet's Syndrome; vasculitissarcoidosis amyloidosis; allergies; anaphylaxis; systemic mastocytosis;and infectious diseases of the internal organs such as hepatitis orulcers.

Typically, a K6 modulator is administered to a mammal such as a humanpatient that has been diagnosed with an inflammatory condition (e.g.,MS). Suitable modulators can decrease the expression of a nucleic acidencoding K6, decrease levels of the K6 protein, or inhibit K6 activity.K6 modulators that can be used include, for example, antibodies havingspecific binding affinity for K6, antisense K6 molecules, selectiveserine protease inhibitors, and pharmaceutically acceptable saltsthereof. K6 modulators also can be administered prophylactically inpatients at risk for developing inflammatory conditions to preventdevelopment of symptoms of the disease from occurring, delaying onset ofsymptoms, or lessening the severity of subsequently developed diseasesymptoms. As described herein, immunization with K6 in an autoimmunemodel of MS (experimental allergic encephalomyelitis (EAE) model)inhibited the development of clinical signs of EAE. In either case, anamount of a K6 modulator effective to treat the inflammatory conditionis administered to the patient. Treatment of an inflammatory conditioncan include reducing the severity of the disease or slowing progressionof the disease. As used herein, the term “effective amount” refers to anamount of a K6 modulator that reduces the deleterious effects of theinflammatory condition without inducing significant toxicity to thehost. Effective amounts of K6 modulators can be determined by aphysician, taking into account various factors that can modify theaction of drugs such as overall health status, body weight, sex, diet,time and route of administration, other medications, and any otherrelevant clinical factors.

A K6 modulator can be administered by any route, including, withoutlimitation, oral or parenteral routes of administration such asintravenous, intramuscular, intraperitoneal, subcutaneous, intrathecal,intraarterial, nasal, transdermal (e.g., as a patch), or pulmonaryabsorption. A K6 modulator can be formulated as, for example, asolution, suspension, or emulsion with pharmaceutically acceptablecarriers or excipients suitable for the particular route ofadministration, including sterile aqueous or non-aqueous carriers.Aqueous carriers include, without limitation, water, alcohol, saline,and buffered solutions. Examples of non-aqueous carriers include,without limitation, propylene glycol, polyethylene glycol, vegetableoils, and injectable organic esters. Preservatives, flavorings, sugars,and other additives such as antimicrobials, antioxidants, chelatingagents, inert gases, and the like also may be present.

For oral administration, tablets or capsules can be prepared byconventional means with pharmaceutically acceptable excipients such asbinding agents (e.g., pregelatinized maize starch, polyvinylpyrrolidoneor hydroxypropyl methylcellulose); fillers (e.g., lactose,microcrystalline cellulose or calcium hydrogen phosphate); lubricants(e.g. magnesium stearate, talc or silica); disintegrants (e.g., potatostarch or sodium starch glycolate); or wetting agents (e.g., sodiumlauryl sulfate). Tablets can be coated by methods known in the art.Preparations for oral administration can also be formulated to givecontrolled release of the compound.

Nasal preparations can be presented in a liquid form or as a dryproduct. Nebulised aqueous suspensions or solutions can include carriersor excipients to adjust pH and/or tonicity.

In some embodiments, anti-inflammatory agents are administered incombination with a modulator of K6. For example, a non-steroidalanti-inflammatory agent such as acetaminophen, ibuprofen, or nabumetoneor a steroid such as prednisolone can be administered to a subject. Amodulator of K6 also can be administered with an immunomodulator such asinterferon β (e.g., Betaseron® (recombinant interferon β-1β) and Avonex®(recombinant interferon β-1α): glatiramer acetate (Copaxone®) forrelapsing-remitting MS; or Novantrone®.

Methods of the invention can include monitoring the inflammatorycondition to, for example, determine if the inflammatory condition isimproving with treatment. Any method can be used to monitor aninflammatory condition. For example, for multiple sclerosis patients,lower extremity function, upper extremity function, vision, andcognitive function can be monitored. Magnetic resonance imaging (e.g.,fluid-attenuated inversion recovery) can be performed to examine lesionsand to differentiate old lesions from new or active lesions. Evokedpotential tests can be performed to monitor nerve transmission. Forexample, visual evoked potentials can be used to detect optic neuritis.Brain stem auditory evoked potentials can be used to detectabnormalities in patients with demyelinating lesions in the brainstemthat can cause delays in the transmission of sound. Somatosensory evokedpotentials can be used to detect disruptions in the pathways from thearms and legs to the brain at very specific positions of the CNS.Cerebrospinal fluid can be examined for myelin breakdown products,oligoclonal bands, or IgG antibodies (e.g., IgG index). In addition, asdiscussed below, levels of K6 protein or ribonucleic acid (RNA) can bemonitored.

Anti-K6 Antibodies

Antibodies having specific binding affinity for K6 can be used tomodulate K6 (e.g., decrease activity). As used herein, the terms“antibody” or “antibodies” include intact molecules as well as fragmentsthereof that are capable of binding to an epitopic determinant of K6(e.g., human K6). The term “epitope” refers to an antigenic determinanton an antigen to which the paratope of an antibody binds. Epitopicdeterminants usually consist of chemically active surface groupings ofmolecules such as amino acids or sugar side chains, and typically havespecific three-dimensional structural characteristics, as well asspecific charge characteristics. Epitopes generally have at least fivecontiguous amino acids (a continuous epitope), or alternatively can be aset of noncontiguous amino acids that define a particular structure(e.g., a conformational epitope). The terms “antibody” and “antibodies”include polyclonal antibodies, monoclonal antibodies, humanized orchimeric antibodies, single chain Fv antibody fragments, Fab fragments,and F(ab)₂ fragments. Polyclonal antibodies are heterogenous populationsof antibody molecules that are contained in the sera of the immunizedanimals. Monoclonal antibodies are homogeneous populations of antibodiesto a particular epitope of an antigen.

Antibody fragments that have specific binding affinity for K6 can begenerated by known techniques. For example, F(ab′)2 fragments can beproduced by pepsin digestion of the antibody molecule; Fab fragments canbe generated by reducing the disulfide bridges of F(ab′)2 fragments.Alternatively, Fab expression libraries can be constructed. See, forexample, Huse et al., Science, 246:1275 (1989). Once produced,antibodies or fragments thereof are tested for recognition of K6 bystandard immunoassay methods including ELISA techniques,radioimmunoassays, and Western blotting. See, Short Protocols inMolecular Biology, Chapter 11, Green Publishing Associates and JohnWiley & Sons, Edited by Ausubel, F. M et al., 1992.

Antibodies having specific binding affinity for K6 can be producedthrough standard methods. In general, a K6 polypeptide can berecombinantly produced, or can be purified from a biological sample, andused to immunize animals. As used herein, the term “polypeptide” refersto a polypeptide of at least five amino acids in length. To produce arecombinant K6 polypeptide, a nucleic acid sequence encoding a K6polypeptide can be ligated into an expression vector and used totransform a bacterial or eukaryotic host cell. Nucleic acid constructstypically include a regulatory sequence operably linked to a K6 nucleicacid sequence. Regulatory sequences do not typically encode a geneproduct, but instead affect the expression of the nucleic acid sequence.In bacterial systems, a strain of Escherichia coli such as BL-21 can beused. Suitable E. coli vectors include the pGEX series of vectors thatproduce fusion proteins with glutathione S-transferase (GST).Transformed E. coli are typically grown exponentially, then stimulatedwith isopropylthiogalactopyranoside (IPTG) prior to harvesting. Ingeneral, such fusion proteins are soluble and can be purified easilyfrom lysed cells by adsorption to glutathione-agarose beads followed byelution in the presence of free glutathione. The pGEX vectors aredesigned to include thrombin or factor Xa protease cleavage sites sothat the cloned target gene product can be released from the GST moiety.

Mammalian cell lines that stably express a K6 polypeptide can beproduced by using expression vectors with the appropriate controlelements and a selectable marker. For example, the eukaryotic expressionvector pCDNA.3.1+ (Invitrogen, San Diego, Calif.) is suitable forexpression of a K6 polypeptide in, for example, COS cells, Chinesehamster ovary (CHO), or HEK293 cells. Following introduction of theexpression vector by electroporation, DEAE dextran, or other suitablemethod, stable cell lines are selected. Alternatively, K6 can betranscribed and translated in vitro using wheat germ extract or rabbitreticulocyte lysase.

In eukaryotic host cells, a number of viral-based expression systems canbe utilized to express a K6 polypeptide. A nucleic acid encoding a K6polypeptide can be introduced into a SV40, retroviral or vaccinia basedviral vector and used to infect host cells. Alternatively, a nucleicacid encoding a K6 polypeptide can be cloned into, for example, abaculoviral vector and then used to transfect insect cells. For example,the cDNA encoding the sequence for the mature form of K6 can be insertedinto the pBAC3 transfer vector (Novagen, Madison, Wis.) immediately 3′to the enterokinase (EK) recognition sequence of (Asp)4Lys. This resultsin a 44 amino acid synthetic prosequence (ending in the EK recognitionsequence) leading into the amino-terminal Val-Val-His-Gly (SEQ ID NO:1)sequence of the mature form of K6. Expression of K6 in a pBAC3 transfervector can use the BacVector transfection system (Novagen, Madison,Wis.). The Sf9 insect cell line, in conjunction with sf-900 IIserum-free media (Life Technologies, Rockville, Md.), can be used forpreparation of high-titer (i.e., >10⁹ pfu/mL) viral stock. The TN5(High5, Invitrogen Corp., Carlsbad, Calif.) insect cell line can be usedfor production of expressed protein by the viral stock. Recombinant K6protein can be purified in a single step utilizing the His-tag fusionand nickel affinity resin (Ni-NTA). The eluted K6 fraction can be pooledand extensively dialyzed versus 40 mM Tris-HCl, pH 7.5 (or 40 mM sodiumacetate, pH 4.5), using 6-8 kDa molecular mass cutoff dialysis tubing(Spectrum Laboratories, Rancho Dominguez, Calif.).

Various host animals can be immunized by injection of the K6polypeptide. Host animals include rabbits, chickens, mice, guinea pigsand rats. Various adjuvants that can be used to increase theimmunological response depend on the host species and include Freund'sadjuvant (complete and incomplete), mineral gels such as aluminumhydroxide, surface-active substances such as lysolecithin, pluronicpolyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyaninand dinitrophenol. Monoclonal antibodies can be prepared using a K6polypeptide and standard hybridoma technology. In particular, monoclonalantibodies can be obtained by any technique that provides for theproduction of antibody molecules by continuous cell lines in culturesuch as described by Kohler, G. et al., Nature, 256:495 (1975), thehuman B-cell hybridoma technique (Kosbor et al., Immunology Today, 4:72(1983); Cole et al., Proc. Natl. Acad. Sci USA, 80:2026 (1983)), and theEBV-hybridoma technique (Cole et al., “Monoclonal Antibodies and CancerTherapy”, Alan R. Liss, Inc., pp. 77-96 (1983)). Such antibodies can beof any immunoglobulin class including IgG, IgM, IgE, IgA, IgD, and anysubclass thereof. The hybridoma producing the monoclonal antibodies ofthe invention can be cultivated in vitro and in vivo.

In some embodiments, antibodies of the invention can inhibit theenzymatic activity of K6. In vitro assays can be used to monitor K6activity after incubation in the presence of an antibody. Typically, K6can be incubated with an antibody (e.g, polyclonal or monoclonal), thenthe ability of K6 to cleave a substrate such as myelin basic protein oran arginine-specific fluorogenic substrate can be assessed at 37° C. ina suitable buffer (e.g., Tris buffer). Depending on the substrate,cleavage can be monitored using sodium dodecyl sulfate(SDS)-polyacrylamide gel electrophoresis (PAGE) or a spectrophotometer.

Antisense Oligonucleotides

Antisense oligonucleotides can be used to modulate K6 by decreasinglevels of K6 protein. The antisense oligonucleotides in accordance withthis invention are at least 8 nucleotides in length. For example, anucleic acid can be about 8, 9, 10-20 (e.g., 11, 12, 13, 14, 15, 16, 17,18, 19, or 20 nucleotides in length), 15 to 20, 18-25, or 20-50nucleotides in length. In other embodiments, antisense molecules can beused that are greater than 50 nucleotides in length, including thefull-length sequence of a K6 mRNA. As used herein, the term“oligonucleotide” refers to an oligomer or polymer of ribonucleic acid(RNA) or deoxyribonucleic acid (DNA) or analogs thereof. Nucleic acidanalogs can be modified at the base moiety, sugar moiety, or phosphatebackbone to improve, for example, stability, hybridization, orsolubility of a nucleic acid. Modifications at the base moiety includesubstitution of deoxyuridine for deoxythymidine, and5-methyl-2′-deoxycytidine and 5-bromo-2′-deoxycytidine fordeoxycytidine. Other examples of nucleobases that can be substituted fora natural base include 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and otheralkyl derivatives of adenine and guanine, 2-propyl and other alkylderivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil andcytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil),4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl andother 8-substituted adenines and guanines, 5-halo particularly 5-bromo,5-trifluoromethyl and other 5-substituted uracils and cytosines,7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine,7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine.Other useful nucleobases include those disclosed, for example, in U.S.Pat. No. 3,687,808.

Modifications of the sugar moiety can include modification of the 2′hydroxyl of the ribose sugar to form 2′-O-methyl or 2′-O-allyl sugars.The deoxyribose phosphate backbone can be modified to produce morpholinonucleic acids, in which each base moiety is linked to a six-membered,morpholino ring, or peptide nucleic acids, in which the deoxyphosphatebackbone is replaced by a pseudopeptide backbone (e.g., anaminoethylglycine backbone) and the four bases are retained. See, forexample, Summerton and Weller (1997) Antisense Nucleic Acid Drug Dev.7:187-195; and Hyrup et al. (1996) Bioorgan. Med. Chem. 4:5-23. Inaddition, the deoxyphosphate backbone can be replaced with, for example,a phosphorothioate or phosphorodithioate backbone, a phosphoroamidite,or an alkyl phosphotriester backbone. See, for example, U.S. Pat. Nos.4,469,863, 5,235,033, 5,750,666, and 5,596,086 for methods of preparingoligonucleotides with modified backbones.

Antisense oligonucleotides of the invention also can be modified bychemical linkage to one or more moieties or conjugates that enhance theactivity, cellular distribution or cellular uptake of theoligonucleotide. Such moieties include but are not limited to lipidmoieties (e.g., a cholesterol moiety); cholic acid; a thioether moiety(e.g., hexyl-S-tritylthiol); a thiocholesterol moiety; an aliphaticchain (e.g., dodecandiol or undecyl residues); a phospholipid moiety(e.g., di-hexadecyl-rac-glycerol or triethyl-ammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate); a polyamine or apolyethylene glycol chain; adamantane acetic acid; a palmityl moiety; oran octadecylamine or hexylamino-carbonyl-oxycholesterol moiety. Thepreparation of such oligonucleotide conjugates is disclosed in, forexample, U.S. Pat. Nos. 5,218,105 and 5,214,136.

Methods for synthesizing antisense oligonucleotides are known, includingsolid phase synthesis techniques. Equipment for such synthesis iscommercially available from several vendors including, for example,Applied Biosystems (Foster City, Calif.). Alternatively, expressionvectors that contain a regulatory element that directs production of anantisense transcript can be used to produce antisense molecules.

Antisense oligonucleotides can bind to a nucleic acid encoding K6,including DNA encoding K6 RNA (including pre-mRNA and mRNA) transcribedfrom such DNA, and also cDNA derived from such RNA, under physiologicalconditions (i.e., physiological pH and ionic strength). The nucleic acidsequence encoding human K6 can be found in GenBank under Accession Nos.AF013988, AF149289, and D78203. The nucleic acid sequence encoding ratK6 can be found in GenBank under Accession No. AF016269. For example, anantisense oligonucleotide can hybridize under physiological conditionsto the nucleotide sequence set forth in GenBank Accession Nos. AF013988(FIG. 25; SEQ ID NO:2), AF149289, D78203, or AF016269.

It is understood in the art that the sequence of an antisenseoligonucleotide need not be 100% complementary to that of its targetnucleic acid to be hybridizable under physiological conditions.Antisense oligonucleotides hybridize under physiological conditions whenbinding of the oligonucleotide to the K6 nucleic acid interferes withthe normal function of the K6 nucleic acid, and non-specific binding tonon-target sequences is minimal.

Target sites for K6 antisense oligonucleotides include the regionsencompassing the translation initiation or termination codon of the openreading frame (ORF) of the gene. In addition, the ORF has been targetedeffectively in antisense technology, as have the 5′ and 3′ untranslatedregions. Furthermore, antisense oligonucleotides have been successfullydirected at intron regions and intron-exon junction regions. Furthercriteria can be applied to the design of antisense oligonucleotides.Such criteria are well known in the art, and are widely used, forexample, in the design of oligonucleotide primers. These criteriainclude the lack of predicted secondary structure of a potentialantisense oligonucleotide, an appropriate G and C nucleotide content(e.g., approximately 50%), and the absence of sequence motifs such assingle nucleotide repeats (e.g., GGGG runs). The effectiveness ofantisense oligonucleotides at modulating expression of a K6 nucleic acidcan be evaluated by measuring levels of the K6 mRNA or protein (e.g., byNorthern blotting, RT-PCR, Western blotting, ELISA, orimmunohistochemical staining).

Identifying Modulators of K6

The invention provides methods for identifying K6 modulators that aresuitable for treating one or more inflammatory conditions in mammals. Invitro or in vivo models of inflammatory conditions can be used toidentify suitable modulators of K6. In vitro cell lines, including CG4OLG cell line, or cultured explants or cultures (e.g., purified culturesof OLG progenitors) from an animal model, can be used to identifysuitable K6 modulators. Such cells can be treated with a test compoundover a period of time (e.g., days, weeks, or longer) then samples (e.g.,cells and cell medium) can be collected and examined, for example, forOLG process stability and outgrowth. As a control, the effect of thetest compound can be compared with cultures treated with a serineprotease inhibitor (positive control) and to untreated cultures(negative control). Other assays for identifying K6 modulators includecontacting immune cell cultures with a test compound and determiningtransmigration ability of the cells in vitro. See Example 16 for such anassay. In addition, K6 can be incubated with a test compound and abilityto cleave a substrate can be monitored. See Example 15 for such anassay.

Once a test compound is determined to be effective in vitro, the testcompound can be tested in vivo. For example, a test compound can beadministered to an animal model of multiple sclerosis, such as theTheiler's murine encephalomyelitis virus (TMEV) model or the EAE model(autoimmune model). Samples (e.g., cerebrospinal fluid, blood, serum, ortissue) can be collected over a period of time and assayed. For example,cerebrospinal fluid can be assayed for myelin breakdown products. Spinalcord pathology can be examined for the degree of demyelination andinflammation. Animals also can be examined for behavioral deficits.

The invention provides methods for designing, modeling, and identifyingcompounds that can bind to K6 and inhibit K6 activity. Such compoundsalso can be referred to as “ligands” or “inhibitors.” Compoundsdesigned, modeled, and identified by methods of the invention typicallyhave a binding affinity of at least 1 μM (e.g., at least 500 nM, atleast 100 nM, at least 50 nM, or at least 10 nM) for K6.

Compounds identified by methods of the invention can be polypeptidessuch as, for example, serine protease inhibitors or antibodies.Alternatively, a compound can be any suitable type of molecule that canspecifically bind to K6.

By “modeling” is meant quantitative and/or qualitative analysis ofK6-inhibitor structure/function based on three-dimensional structuralinformation and K6-inhibitor interaction models. This includesconventional numeric-based molecular dynamic and energy minimizationmodels, interactive computer graphic models, modified molecularmechanics models, distance geometry and other structure-based constraintmodels. Modeling typically is performed using a computer and may befurther optimized using known methods.

Methods of designing ligands that bind specifically (i.e., with highaffinity) to K6 typically are computer-based, and involve the use of acomputer having a program capable of generating an atomic model.Computer programs that use X-ray crystallography data are particularlyuseful for designing ligands that can interact with K6. Programs such asRasMol, for example, can be used to generate a three-dimensional modelof K6 and/or determine the structures involved in ligand binding.Computer programs such as INSIGHT (Accelrys, Burlington, Mass.), GRASP(Anthony Nicholls, Columbia University), Dock (Molecular DesignInstitute, University of California at San Francisco), and Auto-Dock(Accelrys) allow for further manipulation and the ability to introducenew structures.

Methods of the invention can include, for example, providing to acomputer the atomic structural coordinates for amino acid residueswithin K6 or a portion of K6, using the computer to generate an atomicmodel of K6 or a portion of K6, further providing the atomic structuralcoordinates of a candidate compound and generating an atomic model ofthe compound optimally positioned to interact with K6, and identifyingthe candidate compound as a ligand of interest if the compound interactswith K6. By “optimally positioned” is meant positioned to optimizehydrophobic interactions between the candidate compound and K6.

Alternatively, a method for designing a ligand having specific bindingaffinity for K6 can utilize a computer with an atomic model stored inits memory. The atomic coordinates of a candidate compound then can beprovided to the computer, and an atomic model of the candidate compoundoptimally positioned can be generated.

Compounds of the invention also may be interactively designed fromstructural information of the compounds described herein using otherstructure-based design/modeling techniques (see, e.g., Jackson (1997)Seminars in Oncology 24:L164-172; and Jones et al. (1996) J. Med. Chem.39:904-917).

Compounds and polypeptides of the invention also can be identified by,for example, identifying candidate compounds by computer modeling asinteracting spatially and preferentially (i.e., with high affinity) withK6, and then screening those compounds in vitro or in vivo for theability to reduce K6 activity or decrease inflammation and/ordemyelination. Suitable methods for such in vitro and in vivo screeninginclude those described herein.

Methods of Using K6 as a Marker for Inflammatory Conditions

Levels of K6 protein or RNA can be used to monitor therapy ofinflammatory conditions, screen for the presence of an inflammatorycondition, or to monitor the disease state (e.g., relapses of MS). Ingeneral, methods of the invention include detecting the level of K6protein or a RNA encoding K6 in a biological sample from a patient(e.g., a human patient) and comparing the level of K6 protein or RNA tothat from a control population (e.g., the average level of K6 from aplurality of subjects without an inflammatory condition). Methods fordetecting levels of K6 protein and RNA are described below. Suitablebiological samples for measuring K6 levels include, for example, blood(including whole blood, plasma, and serum), urine, and cerebrospinalfluid (CSF). Serum and CSF are particularly useful biological samples.

The presence of an inflammatory condition can be determined based on thelevel of K6 protein or RNA relative to the control population. Thus, itis determined if K6 protein or RNA levels are increased, decreased, orthe same as that of the control population. An increase in K6 levelsrelative to that of the control population is indicative of aninflammatory condition. Additional factors that can be considered whendiagnosing an inflammatory condition include, for example, patienthistory, family history, genetic factors, and/or altered neurologicexamination (e.g., for MS or other neurological inflammatory condition).

The levels of K6 protein or RNA in a subject also can be used to monitortreatment. Typically, the subject's baseline level of K6 protein or RNAis obtained (e.g., before treatment) and compared to the level of K6 atvarious time points after or between treatments (e.g., one or more days,weeks, or months after treatment). A decrease in K6 protein or RNAlevels relative to the baseline level is indicative of a positiveresponse to treatment. Similarly, disease state in a subject can bemonitored (e.g., for relapse of disease) by comparing levels of K6protein or RNA in the subject to the subject's baseline level of K6protein or RNA.

Detecting K6 Protein

K6 can be detected, for example, immunologically using one or moreantibodies. In immunological assays, an antibody having specific bindingaffinity for K6 or a secondary antibody that binds to such an antibodycan be labeled, either directly or indirectly. Suitable labels include,without limitation, radionuclides (e.g., ¹²⁵I, ¹³¹I, ³⁵S, ³H, ³²P, ³³P,or ¹⁴C), fluorescent moieties (e.g., fluorescein, FITC, PerCP,rhodamine, or PE), luminescent moieties (e.g., Qdot™ nanoparticlessupplied by the Quantum Dot Corporation, Palo Alto, Calif.), compoundsthat absorb light of a defined wavelength, or enzymes (e.g., alkalinephosphatase or horseradish peroxidase). Antibodies can be indirectlylabeled by conjugation with biotin then detected with avidin orstreptavidin labeled with a molecule described above. Methods ofdetecting or quantifying a label depend on the nature of the label andare known in the art. Examples of detectors include, without limitation,x-ray film, radioactivity counters, scintillation counters,spectrophotometers, colorimeters, fluorometers, luminometers, anddensitometers. Combinations of these approaches (including “multi-layer”assays) familiar to those in the art can be used to enhance thesensitivity of assays.

Immunological assays for detecting K6 can be performed in a variety ofknown formats, including sandwich assays, competition assays(competitive RIA), or bridge immunoassays. See, for example, U.S. Pat.Nos. 5,296,347; 4,233,402; 4,098,876; and 4,034,074. Methods ofdetecting K6 generally include contacting a biological sample with anantibody that binds to K6 and detecting binding of K6 to the antibody.For example, an antibody having specific binding affinity for K6 can beimmobilized on a solid substrate by any of a variety of methods known inthe art and then exposed to the biological sample. Binding of K6 to theantibody on the solid substrate can be detected by exploiting thephenomenon of surface plasmon resonance, which results in a change inthe intensity of surface plasmon resonance upon binding that can bedetected qualitatively or quantitatively by an appropriate instrument,e.g., a Biacore apparatus (Biacore International AB, Rapsgatan, Sweden).Alternatively, the antibody can be labeled and detected as describedabove. A standard curve using known quantities of K6 can be generated toaid in the quantitation of K6 levels.

In other embodiments, a “sandwich” assay in which a capture antibody isimmobilized on a solid substrate is used to detect the level of K6. Thesolid substrate can be contacted with the biological sample such thatany K6 in the sample can bind to the immobilized antibody. The level ofK6 bound to the antibody can be determined using a “detection” antibodyhaving specific binding affinity for K6 and the methods described above.It is understood that in these sandwich assays, the capture antibodyshould not bind to the same epitope (or range of epitopes in the case ofa polyclonal antibody) as the detection antibody. Thus, if a monoclonalantibody is used as a capture antibody, the detection antibody can beanother monoclonal antibody that binds to an epitope that is eithercompletely physically separated from or only partially overlaps with theepitope to which the capture monoclonal antibody binds, or a polyclonalantibody that binds to epitopes other than or in addition to that towhich the capture monoclonal antibody binds. If a polyclonal antibody isused as a capture antibody, the detection antibody can be either amonoclonal antibody that binds to an epitope that is either completelyphysically separated from or partially overlaps with any of the epitopesto which the capture polyclonal antibody binds, or a polyclonal antibodythat binds to epitopes other than or in addition to that to which thecapture polyclonal antibody binds. Sandwich assays can be performed assandwich ELISA assays, sandwich Western blotting assays, or sandwichimmunomagnetic detection assays.

Suitable solid substrates to which an antibody (e.g., a captureantibody) can be bound include, without limitation, microtiter plates,tubes, membranes such as nylon or nitrocellulose membranes, and beads orparticles (e.g., agarose, cellulose, glass, polystyrene, polyacrylamide,magnetic, or magnetizable beads or particles). Magnetic or magnetizableparticles can be particularly useful when an automated immunoassaysystem is used.

Alternative techniques for detecting K6 include mass-spectrophotometrictechniques such as electrospray ionization (ESI), and matrix-assistedlaser desorption-ionization (MALDI). See, for example, Gevaert et al.,Electrophoresis 22(9):1645-51, 2001; Chaurand et al., J Am Soc MassSpectrom 10(2):91-103, 1999. Mass spectrometers useful for suchapplications are available from Applied Biosystems (Foster City,Calif.); Bruker Daltronics (Billerica, Mass.) and Amersham Pharmacia(Sunnyvale, Calif.).

Detecting K6 Ribonucleic Acid

K6 RNA can be detected, for example, by polymerase chain reaction (PCR)assays or RNA blotting techniques (e.g., Northern blotting). Forexample, K6 RNA can be detected in peripheral blood mononuclear cells.In general, PCR refers to amplification of a target nucleic acid, usingsequence information from the ends of the region of interest or beyondto design oligonucleotide primers that are identical or similar insequence to opposite strands of the template to be amplified. Primersare typically 14 to 40 nucleotides in length, but can range from 10nucleotides to hundreds of nucleotides in length. PCR is described, forexample in PCR Primer: A Laboratory Manual. Ed. by Dieffenbach, C. andDveksler, G., Cold Spring Harbor Laboratory Press, 1995. Nucleic acidsalso can be amplified by ligase chain reaction, strand displacementamplification, self-sustained sequence replication or nucleic acidsequence-based amplification. See, for example, Lewis, R., GeneticEngineering News, 12(9):1 (1992); Guatelli et al., Proc. Natl. Acad.Sci. USA, 87:1874-1878 (1990); and Weiss, R., Science, 254:1292 (1991).

For example, the levels of K6 mRNA can be detected using reversetranscription-polymerase chain reaction (RT-PCR). Real-time quantitativePCR can be performed using, for example, the ABI PRISM 7700 SequenceDetection System and Taqman fluorogenic probes, or the LightCycler™instrument from Roche.

Articles of Manufacture

Antibodies having specific binding affinity for K6 can be combined withpackaging material and sold as a kit for detecting K6 from biologicalsamples, treating inflammatory conditions, monitoring therapy ofinflammatory conditions, or monitoring disease relapses (e.g., of MS).Antisense oligonucleotides that inhibit expression of K6 also can becombined with packaging material and sold as a kit for treatinginflammatory conditions. Components and methods for producing articlesof manufactures are well known. The articles of manufacture may combineone or more anti-K6 antibodies or fragments thereof or one or moreantisense oligonucleotides as described herein. In addition, thearticles of manufacture may further include reagents such as secondaryantibodies, buffers, indicator molecules, solid phases (e.g., beads),additional anti-inflammatory agents, and/or other useful reagents fordetecting K5 from biological samples, treating inflammatory conditions,monitoring therapy of inflammatory conditions, or monitoring diseaserelapses. The anti-K6 antibody or antisense oligonucleotide can be in acontainer, such as a plastic, polyethylene, polypropylene, ethylene, orpropylene vessel that is either a capped tube or a bottle. In someembodiments, the anti-K6 antibody can be included on a solid phase suchas a handheld device for bedside testing. Instructions describing howthe various reagents are effective for treating inflammatory conditions,monitoring therapy of inflammatory conditions, or monitoring diseaserelapses also may be included in such kits.

The invention will be further described in the following examples, whichdo not limit the scope of the invention described in the claims.

EXAMPLES Materials and Methods

Human multiple sclerosis (MS) lesions: This study was performed onparaffin-embedded and formalin-fixed archival material from autopsieswith clinically and pathologically confirmed MS. Paraffin-embedded 5-μmsections were stained with routine neuropathological stains, includinghematoxylin-eosin, Luxol fast blue/periodic acid Schiff (LFB/PAS), andBileschowski silver impregnation axonal stain, as well asimmunocytochemistry for the following markers: anti-proteolipid protein(MCA839, Serotec, Raleigh, N.C.), anti-myelin oligodendrocyteglycoprotein (Dr. S. Piddlesden, University of Cardiff, UK), and anti-K6(see below). All cases underwent detailed neuropathological examinationand were screened for white matter demyelinating lesions. Demyelinatingactivity was classified according to recently established criteria(Lassmann et al. (1998) J. Neuroimmunol. 86:213-217). Activedemyelinating lesions were diffusely infiltrated by macrophagescontaining myelin proteins as markers of recent and ongoing myelinphagocytosis. Inactive demyelinated lesions were completely demyelinatedwithout signs of remyelination.

Theiler's murine encephalomyelitis virus (TMEV) model of MS: Four- to8-week-old female SJL/J (H-2^(S)) mice (Jackson Laboratories, BarHarbor, Me.) were intracerebrally injected with 2×10⁶ plaque-formingunits (p.f.u.) of the Daniel's strain of TMEV, in a 10 μl volume. Careand handling of the mice was in accord with the guidelines of both theNIH and the Mayo Clinic Animal Care and Use Committee. At 30, 45, 90,120 and 180 days post-infection, mice were anaesthetized withpentobarbital (150 mg/kg) and perfused with 4% paraformaldehyde. Spinalcords were blocked transversely at 1 mm, cryoprotected in 25% sucrose,frozen on dry ice and sectioned transversely at 20 pm. Alternatively,blocks were embedded in paraffin and cut at 5 pm. Unfixed spinal cordswere obtained at the same time points, snap frozen and stored at −70° C.until analysis.

MOG-induced EAE: Marmosets were obtained from Clea, Japan, or the NewEngland Regional Primate Research Center, and housed in the primatecolony at the University of California, San Francisco, according to allguidelines of the Institutional Animal Care and Use Committee. EAE wasinduced by immunization with 100 pg of recombinant rat αMOG(extracellular domain, containing amino acids 1-125), emulsified incomplete Freund's adjuvant (CFA), followed by intravenous injection of10¹⁰ killed Bordetella pertussis organisms on the day of immunizationand 48 h later. Clinical signs of EAE developed between 19 and 23 daysafter immunization. Animals were euthanized with worsening signs duringthe acute phase (40-42 days after immunization), under deep barbiturateanesthesia, by intracardiac perfusion with 4% paraformaldehyde. Slabs ofspinal cord were processed for paraffin and 5-pm thick sections werestained with hematoxylin and eosin, or processed to localize K6immunoreactivity.

Immunohistochemistry: Immunostaining of K6 in mouse, marmoset and humantissue sections was accomplished using purified biotin. Rat conjugatedor unconjugated mouse monoclonal K6 anti-brain antibodies (Scarisbricket al. (2000) Glia 30:219-230) or rabbit polyclonal antibodies (Blaberet al. (2002) Biochem. 41:1165-1173), each of which yielded identicalstaining patterns in the tissues examined. Cell mixture-specific markersused in double-labelling studies were: anti-glial fibrillary acidicprotein (anti-GFAP); Cy3 conjugate (Sigma, St Louis, Mo.); ratanti-mouse F480 IgG (Serotec); biotinylated isolectin B4 (Sigma); ratanti-mouse CD4; or rat biotinylated antiTricine mouse CD8b.2(PharMingen, San Diego, Calif., USA). Bound antibodies were detectedusing mouse adsorbed, fluorochrome-conjugated, species appropriatesecondary antibodies (Jackson ImmunoResearch, West Grove, Pa.), or withthe avidin-biotin immunoperoxidase technique (Vector Laboratories,Burlingame, Calif.). In all cases, control for the specificity ofimmunostaining included staining as above with the omission of primaryantibody.

In situ hybridization: Examination of K6 and proteolipid protein (PLP)mRNA expression in TMEV-infected mouse spinal cord was accomplishedusing digoxigenin (DIG)-labelled cRNA probes. The K6-specific probe wasprepared by transcription from the K6 cDNA construct pM514, containing435 base pairs (bp) of rat K6 (nucleotides 220-655), and the PLP probefrom construct pGPLP-1, containing 250 bp of mouse PLP (nucleotides34-285). Hybridization was performed as described previously(Scarisbrick et al. (1999) J. Neurosci. 19:7757-7769), and in some caseshybridized slides were further processed to localize K6-IR, GFAP-IR orIsolectin _(B4)-IR, as detailed above.

Recombinant K6: Recombinant rat K6 (r-K6) was expressed in thebaculovirus system, as described in detail elsewhere (Blaber et al.supra). Briefly, the zymogen form of r-K6 with a 44 amino acid syntheticpro-sequence including an enterokinase (EK) recognition sequence and 6×histidine tag, was expressed in baculovirus expression system, purifiedin a single step utilizing the His tag fusion and nickel affinity resinand shown to be 98% pure by Coomassie Blue-stained SDS-PAGE. Thehomogeneity of purified K6 was confirmed using N-terminal sequencing,mass spectrometry and size exclusion high-performance liquidchromatography. After activation by EK (Roche Diagnostics Corp.,Indianapolis, Ind.), mature r-K6 was further purified by G-50 superfine(Pharmacia Corp., Kalamazoo, Mich.) size exclusion chromatography, toeliminate enterokinase and the cleaved propeptide.

Degradation of myelin basic protein and MOG by K6: Rat myelin basicprotein (MBP) was isolated from adult rat and was incubated with r-K6 in40 mM phosphate, 150 mM NaCl (pH 7.4) at a 100:1 mass ratio. The finalconcentration of r-K6 in the reaction was 35.3 pM. The reaction mixturewas incubated at 37° C., time points were taken at 0, 1, 4, and 16 hourspost-monoclonal incubation, snap frozen on dry ice and kept at −80° C.The digestion pattern of rat MBP was analyzed by loading 10 pl of sample(equivalent to 5 pg of rat MBP) per lane on 16.5% SDS-PAGE underreducing conditions.

Recombinant rat myelin oligodendrocyte glycoprotein (αMOG), as describedabove, was incubated with r-K6 in the same conditions as rat MBP, exceptthat the final concentration of αMOG was 31.5 pM. The digested samplewas resolved on 16.5% Tricine SDS-PAGE for analysis in the same manner.

Oligodendrocyte cell culture systems: Two oligodendrocyte culturesystems were used: purified oligodendrocyte progenitors and thebipotential CG4 oligodendrocyte cell line. Mixed primary glial cellcultures were prepared from the telencephala of PN-1 Sprague-Dawleyrats, and OL progenitors obtained from these by overnight shaking anddifferential adhesion as described in detail in McCarthy and De Vellis((1980) J. Cell. Biol. 85:890-902). Purified OL progenitors were platedonto poly-L-ornithine coated glass coverslips at a density of 20×10³/cm²and grown in Dulbecco's minimal essential media (DMEM) containing: 4.5mg/ml glucose, 2 mM glutamine, N2 supplement (Gibco-BRL, Grand Island,N.Y.), 5 μg/ml insulin, 30 nM T3, 10 ng/ml biotin, 50 U/mlpenicillin/streptomycin, 0.1 mg/ml sodium pyruvate (Sigma), and 10 ng/mleach of PDGF-AA and bFGF, (R &D Systems, Minneapolis, Minn.).Undifferentiated CG4 cells were grown in Ham's DMEM F12 containing thesame supplements.

To examine the effects of excess exogenous r-K6, telencephalon-derivedor CG4 O2A progenitor cells were differentiated toward theoligodendrocyte lineage by replacement of mitogenic factors, PDGF andFGF, with 0.05% bovine serum albumin (BSA). The effect of r-K6 ondifferentiated oligodendrocytes was examined by exposing cellsdifferentiated for 72 hours to 1 or 10 μg/ml (40 or 400 nM) of r-K6 foran additional 72 hours, with media changes containing fresh r-K6 every24 hours. To evaluate the effect of r-K6 on oligodendrocytedifferentiation, progenitors were plated in differentiation media asabove, but media was supplemented with 1 or 10 μg/ml of r-K6 after a 30minute culture period, allowing for cellular attachment. As above, cellswere then allowed to differentiate for a further 72 hours beforeanalysis. To distinguish between cell surface or substrate effects, in athird paradigm, CG4 O2A cells were treated in one of three ways: (i)cells were plated and media changed to differentiation media containing1 or 10 μg/ml of r-K6 30 minutes after plating; (ii) cells wereresuspended in and incubated for 30 minutes with 1 or 10 μg/ml of r-K6,spun down and resuspended in protease-free differentiation media beforeplating; or (iii) prior to plating, the polyornithine coated coverslipwas incubated for 1 hour at 37° C. with 1 or 10 μg/ml of r-K6. Cellswere differentiated for a further 24 hours prior to analysis. Controlwells were supplemented with an equal volume of vehicle (40 mM NaOAc,100 mM NaCl, pH 4.5) alone. To visualize oligodendrocyte processes,coverslips were briefly rinsed in HEPES-buffered saline solution (HBSS),and stained live in HBSS containing 1% BSA for the presence of cellsurface sulphatide, using the monoclonal antibody, and fluorescein(FITC)-conjugated secondary antibodies (Jackson ImmunoResearch).Labelled cells were fixed in 2% paraformaldehyde and coverslipped with90% glycerol (pH 8.0) containing 10 μg/ml of the nuclear stainbisbenzamide (Sigma).

In each cell culture paradigm, process outgrowth and cell number wereevaluated from six (165 mm²) fields per coverslip, which were imageddigitally (40× objective) using an Olympus AX70 microscope fitted with aSPOT color digital camera (Diagnostic Instruments, Inc., SterlingHeights, Mich.). The number of oligodendrocyte O4-immunoreactiveprocesses that crossed horizontal lines of a 0.25 inch grid superimposedon each image were counted for each field. Additionally, in each fieldcounts were also made of the total number of O4-positive cells and allcells stained with bisbenzamide. On average, 120 cells were counted perculture condition in each experiment. The mean and standard error ofcounts from triplicate wells were calculated and analyzed by one-wayanalysis of variance and the Student Newman Keuls (SNK) post hoc test.All experiments were performed in triplicate and repeated at least twiceusing independent cell culture preparations.

Overexpression of K6 in CG4 cells: To prepare the green fluorescentprotein (GFP)-rat K6 construct, the full-length K6 clone, without a stopcodon, was amplified by polymerase chain reaction from vector SB12-42B,and subcloned in-frame with the cycle 3 GFP protein ofpcDNA3.1/CT-GFP-TOP0 (Invitrogen, Carlsbad, Calif.). For cellulartransfection, vectors containing the K6-GFP construct, or GFP alone,were digested with BglII, ethanol precipitated and resuspended insterile water. Proliferating CG4 cells grown on polyornithine-coated 60mm dishes at a density of 2×10⁵ per 35 mm well were transfected with 2μg of DNA, using FuGENE 6 reagent (Roche Diagnostics). Cellssuccessfully transfected were identified by expression of cycle 3 GFPwhen viewed with a 40× objective on an inverted Olympus IX70 microscopefitted with a FITC filter set. GFP-positive cells were imaged digitallyusing both fluorescence and phase microscopy, at 24, 48 and 96 hourspost-transfection, and the number of processes was evaluated by countingthose that crossed a superimposed 0.25 inch grid. At each time point thenumber of processes associated with cells transfected with K6-GFP or GFPalone were compared using the Mann-Whitney rank sum test.

Expression and crystallization of K6 and data collection: Mature activehuman K6 (hK6) was expressed and purified from a baculovirus/insect cellline system essentially as described for the rat K6 homolog. PurifiedhK6 was concentrated to 20 mg/mL in 40 mM sodium acetate, 100 mM NaCl,and 20 mM benzamidine, pH 4.5. Crystallization conditions wereidentified using a hanging-drop sparse-matrix screen of precipitants,salts, and pH conditions (Hampton Research, Laguna Niguel, Calif.).Diffraction quality crystals grew from 30% (w/v) PEG 4000, 0.2 Mmagnesium chloride hexahydrate, and 0.1 M Tris hydrochloride, pH 8.5after two weeks incubation at 4° C. X-ray intensity data were collectedat 103 K from a single crystal (0.5×0.2×0.05 mm) with a Rigaku imagingplate area detector R-Axis IIc using Cu—Kα radiation. Data wereprocessed and scaled using DENZO and SCALEPACK 27,28. This crystaldiffracted to at least 1.75 Å. The space group was tentativelyidentified as orthorhombic P212121 with cell constants a=39.1 Å, b=62.1Å, c=85.8 Å. Based upon a molecular mass of approximately 29 kDA forhK6, a Matthews coefficient Vm=1.80 Å3/Da suggested a single molecule inthe asymmetric unit 29.

Molecular Replacement and Structure Refinement: Initial phases werecalculated by molecular-replacement using Atlantic salmon trypsin (PDBcode 1A0J) as a search model and the Crystallography & NMR System (CNS)software package 30. The rotational search resulted in a single peak 8σabove the noise level, and a subsequent translational search in theP212121 space group of the correctly rotated model resulted in a singlepeak 4σ above the noise level. The Rcryst was 47.3% after rigid bodyrefinement of this initial solution. A 3 Å 2Fobs—Fcalc SIGMAA-weightedcomposite annealed omit map (5% of data omitted) was calculated and thestructure was built and refined through alternating cycles using thegraphic program O 31 and CNS. All refinements were performed bysimulated annealing using a maximum likelihood target, and this cyclicprocedure was repeated several times with gradual increase of theresolution to 1.75 Å. A random selection of 3% of the data was assignedfor calculation of Rfree and was not included in the refinement. Solventmolecules were added at the last stage of refinement at stereochemicallyreasonable positions.

Generation of hK6 Specific Monoclonal Antibodies—Monoclonal antibodiesrecognizing K6 were generated using human recombinant K6 expressed inthe baculovirus system. Enterokinase activated hK6 was emulsified inequal ratios of Complete Freund's Adjuvant (CFA) and 0.01M phosphatebuffered saline (PBS), to a final concentration of 500 μg per ml. Balb/cmice (Jackson Laboratories, Bar Harbor, Me.), were immunized in at twosites subcutaneously, and at the base of the tail, with a total of 200μl of the CFA emulsion. After 30 days, each mouse was given asubcutaneous boost of 50 μg hK6 dissolved in 100 μl PBS. Six days afterthe boost, splenic lymphocytes were fused with myeloma cells. Theprotocol used for generation of B cell hybridomas followed standardprocedures (Faedas de St. Groth and Scheidegger, 1980). Briefly, spleenswere removed from mice and single-cell suspensions were prepared withred blood cells lysed with ammonium chloride buffer. Splenic lymphocytesand F/O meyeloma cells (non-secreting myeloma derived from sp2/0 balb/cmyeloma cells), were mixed at a 5:1 ratio and centrifuged to form apellet. The cell pellet was resuspended in 1 ml of a 50% solution ofpolyethylene glycol 1540, then incubated at 37° C. for 90 seconds. Cellswere washed and resuspended in fresh media, and 100 μl aliquots wereadded to the wells of microtiter plates. After 24 hrs, 100 μl culturemedium supplemented with 1 M hypoxanthine (HT), 4 mM aminopterin, and0.16 M thymidine (HAT), were added to each well. Every 3-4 daysthereafter, 100 μl culture medium was replaced with 100 μl fresh mediumcontaining HAT, HT, and complete medium without HAT, successively over aperiod of approximately 14 days. Upon reaching 75% confluency theculture supernatants were screened by ELISA for the presence K6-specificantibody, using recombinant hK6 coated plates Immunolon II plates orrecombinant human kallikrein 3 (hK3) coated plates, as a negativecontrol using standard techniques. K6-specific hybridomas were thencloned in limiting dilution cultures at 1 cell per microtiter well.Balb/c spleen cells served as feeder layer cells for fusion and cloningmicrotiter plates (3×10⁵ per well). Fused cells were grown in 96 wellplates in Iscove's Modified Dulbeco's Medium, containing 10.0% FetalBovine Serum. The supernatants of these clones were screened forK6-reactivity by ELISA (as above), and K6-specific clones were subclonedat 0.3 cells per microtiter well. The supernatants of subclones werescreened by ELISA for K6-reactivity and selected subclones expanded.K6-specific hybridomas were grown in roller bottles in serum free mediaand the IgG fraction purified using Protein G (Pharmacia). Purifiedantibodies were isotyped as IgG or IgM using a mouse monoclonal antibodyisotyping kit (IsoStrip, Boehringer Mannheim, Indianapolis, Ind.). Twomonoclonal generated in this fashion, K6-1 and K6-2, are IgG-1_(K)specific and recognize both rodent and human recombinant K6. Unlessotherwise specified all reagents were obtained from Sigma (St. Louis,Mo.).

Example 1 K6 is Abundantly and Differentially Expressed in the AdultHuman CNS

The level of K6 mRNA in adult human brain and peripheral tissues wasdetermined. Radiolabeled human K6 cDNAs were hybridized to a dot blotcontaining human RNA from 43 different regions, including the brain(rows A and B) and peripheral tissues (rows C to F). The highest levelsof K6 mRNA were present in the spinal cord (B7) and medulla oblongata(A8) (see Table 1). In contrast, the level of K6 mRNA in samples of mostperipheral tissues was low, with the exception of the kidney (E1), wherethe level of K6 mRNA detected was similar to that detected in the cord(B7). Low levels of K6 mRNA also were present in human thymus (E5),spleen (E4) and lymph node (E7).

TABLE 1 Quantification of K6 mRNA present in human brain regions andperipheral tissues. RNA Source K6 A1 - Whole Brain 18* A2 - Arnygdala 15A3 - Caudate Nucleus 19 A4 - Cerebellum 8 A5 - Cerebral Cortex 20 A6 -Frontal Lobe 43 A7 - Hippocanipus 57 A8 - Medulla Oblongata 102 B1 -Occipital Pole 9 B2 - Putamen 21 B3 - Substantia Nigra 33 B4 - TemporalLobe 13 B5 - Thalamus 29 B6 - Sub-thalamic Nucleus 34 B7 - Spinal Cord100 C1 - Heart 7 C2 - Aorta 5 C3 - Skeletal Muscle 9 C4 - Colon 12 C5 -Bladder 12 C6 - Uterus 13 C7 - Prostate 13 C8 - Stomach 16 D1 - Testis13 D2 - Ovary 9 D3 - Pancreas 9 D4 - Pituitary Gland 16 D5 - AdrenalGland 16 D6 - Thyroid Gland 19 D7 - Salivary Gland 14 D8 - Mammary Gland17 E1 - Kidney 87 E2 - Liver 23 E3 - Small Intestine 15 E4 - Spleen 9E5 - Thymus 23 E6 - Peripheral Leukocyte 14 E7 - Lymph Node 17 E8 - BoneMarrow 15 F1 - Appendix 9 F2 - Lung 20 F3 - Trachea 9 F4 - Placenta 9*The relative optical density produced by hybridization of a human K6cDNA probe to each RNA sample was expressed as percent of the signalproduced in the spinal cord.

Example 2 K6 is Robustly Expressed by Oligodendrocytes

Oligodendrocytes in all human brain regions were examined byimmunohistochemistry as described in the Methods and Materials section,and were found to be densely K6-immunoreactive. The immunoreactiveoligodendrocytes included those of the corpus callosum, the optic nerve,and the subcortical white matter. The localization of K6 tooligodendrocytes, but not to astrocytes, in white matter of the adultbrain suggests this enzyme plays a role in normal oligodendrocytehomeostatic mechanisms.

Example 3 The Expression of K6 by Oliodendrocytes is Upregulated byInjury

Levels of K6 mRNA in adult rat spinal cord were increased in both whiteand gray matter following kainic acid (KA)-induced excitotoxic injury.Dark-field photomicrographs (FIG. 1) show the autoradiographiclocalization of K6 mRNA in a transverse section through the spinal cordof a control rat, and in parallel sections of paired experimental ratsat 3 and 7 days after intraperitoneal injection of KA. By three daysafter KA treatment, K6 mRNA was 2-fold higher in the white matter and1.5-fold higher in the dorsal horn (DH) and ventral horn (VH) of thespinal cord gray matter, compared to controls (P<0.05). A hemisection ofspinal cord white matter from an adult rat revealed a 5-fold increase inK6 mRNA expression by oligodendrocytes by 72 hr-post lesion compared tocontrols. These findings support the hypothesis that K6 is involved inthe response of oligodendrocytes to injury, and suggest that K6 mayparticipate in myelin turnover.

Example 4 K6 is Elevated at Sites of Neuroinflammation and Demyelination

K6 immunoreactivity was dense in oligodendrocytes in areas of normalwhite and gray matter in the spinal cord of control animals (FIG. 2A),and in association with inflammatory cells at sites of activedemyelination 180 days following TMEV infection (FIG. 2B).

A section of spinal cord from a marmoset with αMOG-induced EAE showedfoci of inflammatory cells, seen as areas of hyper-cellularity inhematoxylin/eosin counter-stained sections. In an adjacent section, K6immunoreactivity was associated with oligodendrocytes in normal whitematter and with inflammatory cells at sites of active demyelination.

Human brain sections from autopsies with clinically and pathologicallyconfirmed MS were stained with LAF/PAS. These studies revealed awell-demarcated chronic active MS lesion (FIG. 3A, shown at a highermagnification in FIG. 3B). The plaque (PL) showed a complete loss ofmyelin compared to the periplaque white matter (PPWMK.Immunohistochemical analysis demonstrated that K6 was upregulated withininflammatory cells at the border between the PL and the PPWM, on theside of the lesion (FIG. 3C, shown at a higher magnification in FIG.3D). The highest levels of K6 immunoreactivity were associated withinflammatory cells at the PL/PPWM border. K6 immunoreactivity also wasupregulated within reactive astrocytes.

Example 5 K6 is Expressed by Macrophages, in Addition to Both CD4 andCD8 Inflammatory Cells at Sites of Demyelination in the TMEV-InfectedMouse Spinal Cord

The earliest known event in the pathogenesis of MS lesions istransendothelial migration of lymphocytes into the CNS. As discussedabove, K6 immunoreactivity in both TMEV and αMOG induced EAE, as well asin active human MS lesions, is dense in inflammatory cells at sites ofdemyelination. The potential role of K6 in demyelinating disease wastherefore examined.

K6 was localized by immunofluorescence at sites of demyelination inmouse spinal cord white matter, 90 days post-infection with TMEV. Thewhite matter sections also were stained with markers for inflammatorycells, including F480, CD4, and CD8. This double-labeling techniquedemonstrated high levels of K6 immunoreactivity associated withmacrophages, as well as CD4 and CD8 T cells. K6 immunoreactivity was notupregulated in oligodendrocytes in areas of demyelination, at leastrelative to the high levels observed in inflammatory cells.

Example 6 K6 is Elevated in the Sera of TMEV Infected Mice

A quantitative capture enzyme-linked immunosorbent assay (ELISA) wasused to determine the amount of K6 in sera from uninfected mice(control), or in mice at 90 and 180 days post-TMEV infection (FIG. 4).There was about a 3.5-fold increase in the amount of K6 in sera at 180days following infection, compared with control sera.

Example 7 Recombinant K6 Degrades Components of the Blood Brain Barrier

To determine whether K6 may participate in the migration of inflammatorycells into the CNS by degrading the BBB basal lamina, enzymatic studiesof K6 with collagen, fibronectin, and laminin were conducted. K6 rapidlydegraded all three BBB components. Collagen type I was degraded within 5minutes of K6 addition (FIG. 5A, lane 3 compared to lane 2). Fibronectinwas partially degraded within 1 hour of K6 addition and was completelydegraded by 24 hours (FIG. 5B, lanes 3 and 4 compared to lane 2).Laminin was partially degraded by one hour after K6 addition, and wascompletely degraded by 16 hours (FIG. 5C, lanes 2 and 3 compared to lane1). K6 therefore may play a role in the migration of inflammatory cellsinto the CNS.

Example 8 Recombinant K6 can Degrade Myelin Specific Proteins

K6 is a trypsin-like serine protease that specifically hydrolysesproteins after Arginine residues. Given this broad substratespecificity, it is likely that increased levels of K6 at sites of CNSinflammation will have widespread degradative functions, including thebreakdown of myelin specific proteins. This hypothesis was tested invitro by examining the ability of K6 to degrade MBP and the 125 aminoterminal fragment of αMOG. MBP was partially degraded by 10 minutesafter addition of K6, and showed much greater levels of degradation at30 minutes and 60 minutes (FIG. 6A, lanes 7-9). αMOG was partiallydegraded by 1 hour after addition of K6, and was completely degraded by4 hours (FIG. 6B, lanes 3 and 4).

Example 9 K6 is Capable of Enhancing the Migration of Inflammatory CellsAcross Blood Brain Barrier ECM

The migration of human peripheral blood mononuclear cells (PBMC) wasassessed. Matrigel-coated 8 mm pore size membranes were pretreated with10 μg/ml recombinant K6 (r-K6) for 24 hr prior to the addition of cells,using fetal calf serum (FCS) as the chemoattractant. An 8 hourincubation period in the presence of a FCS gradient revealed thatpre-treatment of a Matrigel membrane with r-K6 significantly enhancedPBMC migration (FIG. 7; P<0.05).

Example 10 K6 is Important in Normal Oligodendrocyte Development

Two oligodendrocyte culture systems, primary “shaken” oligodendrocytes(McCarthy and De Vellis, supra) and the CG4 oligodendrocyte cell line(Louis et al. (1992) Glia 6:30-38), in addition to sensitiveimmunohistochemical, immunoblot and ELISA assays, were used to show thatK6 is expressed by oligodendrocyte progenitors (O2A) right through tomaturation in vitro. While abundant at all stages of the lineage, thedistribution of K6 was altered with maturation, becoming more abundantin association with distal processes. This dynamic expression patternpoints to roles for K6 in the development and maintenance of the maturemyelinating phenotype.

Using immunohistochemistry, K6 and oligodendrocyte lineage markers wereshown to colocalize in purified cultures of oligodendrocyte progenitorsisolated from the postnatal rat brain, and in the CG4 cell line at theO4 (sulfatide, pro-oligodendrocyte, and O1 (galactocerebroside,immature/mature; stages of differentiation. Both A2B5⁺ (ganglioside)oligodendrocyte progenitors and A2B5⁻ pre-progenitors were K6immunoreactive. It is noted that microglia also were associated withhigh levels of K6 immunoreactivity. In CG4 cells at the O4 and O1 stagesof maturity, K6 immunoreactivity was most abundant in the distalprocesses.

The developmentally regulated expression of K6 in culturedoligodendrocytes was confirmed by immunoblotting (FIG. 8). The highestlevels of K6 (25 kDa) were observed in proliferating oligodendrocytes(Pro), with a progressive decrease as the cell matured in vitro (48hours, 72 hours, and 7 days). Cultured astrocytes also produced K6,albeit at a lower level than oligodendrocytes, but showed a similarprogressive loss of expression as the cells matured. A second band alsowas observed that cross-reacted with the K6 antisera, with a molecularweight of approximately 42 kDa. This band may represent K6 boundcovalently to an endogenous regulator (e.g., an inhibitor).

The putative endogenous inhibitor of K6 appeared to be unique to CNStissues. Western blotting of rat tissue homogenates with rabbitpolyclonal antibodies against r-K6 revealed the presence of both matureK6 and higher molecular weight complexes (FIG. 9). The higher molecularweight complexes, with masses of approximately 42 and 91 kDa, mayrepresent the formation of K6/inhibitor complexes. The larger 91 kDacomplex was observed primarily in the kidney (lane 4), whereas the 42kDa complex was found predominantly in the brain and spinal cord (lanes2 and 3, respectively). A complex of 42 kDa also has been identified inpurified populations of nervous system cells (FIG. 8). These datasupport the presence of unique endogenous K6-inhibitors in neuralcompared with non neural tissues.

Example 11 Excess K6 Results in a Dying Back of OligodendrocyteProcesses

While regulated expression of K6 appears to be important to normaloligodendrocyte development, it is hypothesized that an excess of K6, asis present at sites of CNS inflammation, may contribute tooligodendrogliopathy. To address the potential effect of K6 on thestability of oligodendrocyte processes, oligodendrocyte progenitors wereplated and allowed to differentiate for 72 hours. At this point thewells contained a mixture of O4⁺ and O1⁺ cells. Active r-K6 was added totriplicate wells at a concentration of 1 or 10 μg/ml, and cells wereincubated for an additional 72 hours before staining for sulfatide withan O4 monoclonal antibody. Exposure of mature oligodendrocytes to r-K6resulted in a 2- to 3-fold decrease in the number of O4⁺ processes (FIG.16A), but had no effect on the number of O4⁺ cells (FIG. 16B), or thetotal number of cells (FIG. 16C). Excess K6 thus had a dramatic effecton the stability of oligodendrocyte processes, but did not affect cellsurvival or differentiation toward the O4⁺ lineage (P<0.05).

Over-expression of K6 in oligodendrocyte progenitors also decreasedprocess outgrowth. The full-length K6 clone was inserted into a vectorencoding green fluorescent protein (GFP) to allow production of a K6-GFPfusion protein. CG4 oligodendrocyte progenitors were transfected witheither the K6-GFP construct or the vector encoding GFP alone, and thenumber of processes in cells expressing GFP was determined at 12, 48, or96 hours post-transfection. While neither construct affectedoligodendrocyte survival, cells that over-expressed K6 had significantlyfewer processes from the 48 hour time point onwards, relative to the GFPcontrol (P<0.05). Over-expression of K6 within the cell therefore hadthe same effect as applying K6 exogenously.

Example 12 Inhibition of K6 Attenuates CNS Demyelinating Disease

K6-Inmunization and TMEV Infection: Eight-week old female SJL/J mice(Jackson Laboratories) were each immunized with 100 μg of recombinant K6(r-K6) in 200 μl of complete Freund's Adjuvant (CFA, Difco) containing200 μg of heat-killed mycobacterium. K6 was expressed in the baculovirussystem, purified and activated as described in the Methods and MaterialsSection. Control animals were immunized similarly, receiving thenon-self antigen ovalbumin (OVA, Sigma), CFA alone, or PBS only.Emulsion was injected subcutaneously in each flank, and at the base ofthe tail.

K6 and OVA antibody titers were determined by ELISA, and animals boostedwith an intraperitoneal (or subcutaneous) injection of 25 μg of r-K6, orOVA in PBS, or with PBS alone. One week after the boost, animals wereinjected intracerebrally with 2×10⁶ plaque-forming units (PFU) of theDaniel's strain of TMEV, in a 10 μl volume. Forty days post-TMEVinfection, sections through the spinal cord of each animal wereevaluated histologically, and K6 or OVA antibody titers determined fromsera. Mice were anesthetized with an overdose of sodium pentobarbital(150 mg/kg), and following sera collection, perfused transcardially with4% formaldehyde, 1% glutaraldehyde in PBS (pH 7.4), or 4%paraformaldehyde. Spinal cords were sectioned transversely into 1 mmblocks, and every third block embedded in glycol methacrylate. Forassessment of demyelination, 1 μm thin sections were cut from theplastic embedded blocks, and stained with a modified erichrome stain,with a cresyl violet counterstain. This methodology reproducibly allowsthe visualization of inflammatory cells and the extent of demyelinationwithin the spinal cord.

TMEV DTH: TMEV-specific delayed-type hypersensitivity responses (DTH, ameasure of Th1 immune responses) were determined 48 hr prior to the endof each experiment. Ultraviolet-irradiated purified TMEV (2.5 μg) orPLP139-151 peptide (10 μg) in a 10 μl volume were injected intradermallyinto opposite ears, using a 500 μl Hamilton syringe (Hamilton Co., Reno,Nev.) fitted with a 30-gauge needle. Ear swelling was measured at 24 and48 hr after injection, with a dial gauge micrometer. The mean differencecompared with the pre-injection measurements in each group was comparedbetween K6-immunized mice and control groups.

Quantitative Histology: Quantitative morphologic analysis was performedon 10-15 sections, corresponding to 10-15 different spinal cordsegments, per mouse. Two methods were used to determine the extent ofwhite matter pathology. First, the total white matter area and the totallesion area (in mm²) were calculated using a Zeiss interactive digitalanalysis system (ZIDAS), and camera lucida attached to a Zeissphotomicroscope. Data were expressed as the percent of total lesion areaper total white matter area. Second, a pathologic score reflecting thefrequency of pathology was assigned to each animal based on meningealinflammation and demyelination. The score is expressed as a percentageof the total number of spinal cord quadrants positive for eachpathologic measurement, divided by the total number of spinal cordquadrants examined. A maximum score of 100 reflects the presence ofpathology in all 4 quadrants of every spinal cord segment examined froman individual spinal cord. The significance of differences in thepercent of quadrants with each pathological feature, or in percentlesional area, was determined by One Way Analysis of Variance (ANOVA)and the Student-Newman-Kuel's (SNK) post-hoc test.

The immunization strategy revealed that both inflammation anddemyelination were decreased in the TMEV model of MS. High K6 antibodytiters were observed in the sera of mice 4 weeks after immunization withr-K6 (FIG. 11). The sera of animals immunized with CFA alone did nothave significant K6 antibody titers. Similar results were observed inall immunized mice. Bovine serum albumin (BSA) was substituted for seraas a negative control in this assay.

The percentage of spinal cord quadrants showing demyelination (DM) orinflammation (Infl) was quantified for animals immunized (I) with K6 orCFA alone. Immunization of mice with r-K6 (n=9) 5 weeks prior to TMEVinfection resulted in significantly less DM and Infl in the spinal cordwhen examined at 40 days post-infection, as compared to mice immunizedwith CFA alone (n=10) (P<0.05, FIG. 12). K6-immunization was associatedwith a greater than 3-fold reduction in the percent of the spinal cordwhite matter associated with pathology (ANOVA, P≦0.0023, *SNK, P<0.05).These results were confirmed in two independent experiments.

In CFA-immunized mice, well-formed demyelinated lesions with extensiveperivascular and parenchymal cell infiltrates formed in response to TMEVinfection, but such lesions were significantly less prominent inK6-immunized mice. The amount of demyelination and inflammation wasdetermined in 10 to 15 sections per mouse, by assessing the number ofspinal cord quadrants containing each pathological feature. Spinal cordswere embedded in glycol methacrylate plastic and stained with a modifiederichrome/cresyl violet stain.

Levels of brain pathology were quantified for control animals immunizedwith CFA alone (FIG. 14A) and for animals immunized with K6 (FIG. 14B).A score of 0 reflected the absence of pathology; a 1, no tissuedestruction and minimal inflammation; a 2, early tissue destruction(loss of architecture) and moderate inflammation; a 3, definite tissuedestruction (demyelination, parencymal damage); and a 4 frankparenchymal necrosis. K6 immunized mice had significantly less diseasein all brain regions examined, which included the cerebellum, brainstem, striatum, cortex, hippocampus, corpus callosum, and meningea).

High K6-antibody titers attenuated viral-induced Th-1 response in vivo.DTH responses to the disease-inducing TMEV were evaluated in mice at day38 after infection (n=8 per group). Data shown in FIG. 15 represent themean 48 hr change in ear thickness±SEM, in response to challenge with2.5 μg of UV-irradiated, purified TMEV. DTH responses were significantlyreduced in the K6-immunized mice at the 48 hr time point (*P<0.05,unpaired Students t-test) compared to mice immunized with CFA-containingPBS alone. No significant DTH responses were observed in response toTMEV in mice that were uninfected. No significant DTH responses wereobserved in these mice in response to the self-peptide PLP-139-151 at 38days post-TMEV infection.

Example 13 The Presence of High K6 Antibody Titers, Generated Using anActive Immunization Approach, Decreased Both Spinal Cord Demyelinationand Inflammation, as Well In Vivo and In Vitro Th1 Immune Responses, inan Autoimmune Model of Multiple Sclerosis

Generation of K6 antibody titers and EAE Induction: Function blockingK6-antibodies were generated by active immunization of mice withrecombinant K6 (K6), resulting in the generation of K6-specificantibodies in a self-autonomous fashion. Mice were immunized for K6antibody generation either at the time of EAE induction, using CompleteFreund's Adjuvant, or prior to or EAE induction, using Ribi Adjuvant(Corixa) to avoid repeated CFA administration.

EAE was induced in 12-week old female SJL (H-2^(S)) mice (JacksonLaboratories, Bar Harbor, Mass.) by immunization with 100 μg of thehighly encephalitogenic peptide PLP139-151 (HSLGKWLGHPDKF), dissolved inPBS and emulsified with Incomplete Freund's adjuvant (IFA), containing400 μg of Mycobacterium Tuberculosis, strain H37Ra (Difco, DetroitMich.). In this first series of experiments, mice were immunized with K6at the time of EAE induction, by adding 75 μg per mouse of K6 in PBS, orPBS alone, at the time of emulsification. Groups of 14 mice wereinjected subcutaneously (s.c.) with 0.2 ml of the peptide emulsions, andon the same day, and 48 hr later, injected intraperitoneally (i.p.) with400 ng of Bordetella Pertussis toxin in 0.1 ml of PBS. This high dosePLP139-151 priming protocol produced severe disease with substantialmortality before the 21-day endpoint, precluding meaningful analysis ofpathology. In a second series of experiments therefore, in which theeffects of MSP-pre-immunization were examined, a milder form of EAE waschosen in which consistent clinical disease was induced, but in whichmortality was reduced. High levels of K6 antibody titers wereestablished in these mice prior to the induction of EAE, by immunizingmice with 70 μg of K6 in 100 μl of PBS, mixed with 100 μl of Ribiadjuvant, resuspended in PBS, or Ribi-PBS alone. As a second negativecontrol, other groups of mice received no immunization prior to theinduction of EAE. Two-hundred μl of the Ribi-K6 suspension or Ribialone, were injected s.c. at two sites on the flanks of mice followingthe manufacturers instructions. Sera obtained by tail bleed wereexamined by ELISA to confirm the production of K6 antibodies prior toEAE induction. The lower dose PLP139-151 priming was induced in thesemice by administering 50 μg of PLP 139-151, in IFA containing 200 μg ofM. Tuberculosis, supplemented with 20 μg of K6. Mice were given 100 ngof B. Pertussis toxin on the day of immunization and 48 hr later. Allpeptides were synthesized by the protein core facility at the MayoClinic, with amino acid composition verified by mass spectrometry, andpurity (>98%) confirmed by mass spectroscopy.

Assessment of Clinical Disease: Mice were housed under barrierconditions, and paralyzed mice were afforded easier access to food andwater. The primary end point in these experiments was clinical outcomeand animals were observed daily and graded in a blinded fashionaccording to their clinical severity as follows: grade 0, no clinicaldisease; grade 1, loss of tail tonis; grade 1.5, impairment of rightingreflex; grade 2, paresis or paralysis of one hind limb; grade 3,complete paralysis of both lower extremities; grade 4, non-ambulatoryand moribund; grade 5, death. Sera were collected at the time ofsacrifice to examine K6 and PLP antibody titers.

Statistical Analysis: Where data were non-linear, as in the case ofbehavior scores, the significance of differences between K6-immunizedmice and controls, was determined by non-parametric Mann-Whitney U-test.Parametric unpaired Student's t-test was used for evaluation ofdifferences in histological scores, DTH, T-cell proliferation, cytokineproduction and serum antibody responses, except when data was notnormally distributed in which case the Mann-Whitney U-test was used.Comparison of percent survival between groups was made using X²employing Fisher's exact test. Statistical significance was set atP≦0.05.

Results: K6-immunization at the time of, or prior to PLP-139-151priming, inhibits the development of clinical signs of EAE. The role ofK6 in the development of clinical and histological manifestations of EAEwas investigated by inhibiting K6 enzymatic activity using an activeimmunization approach. The presence of K6-antibodies generated prior to,or at the time of EAE induction, each delayed the onset of disease, andreduced clinical disease scores, in the PLP139-151 EAE model in SJL/Jmice (FIGS. 16 to 19 and Tables 2 and 3).

K6 immunization at the time of PLP139-151 priming was shown to delay themean day of disease onset (day 12.7±0.1 vs. control 10.1±0.4, P=0.006),in addition to the mean time to peak disease (day 14.7±0.5 vs. control13.1±0.54, P<0.04) (FIG. 16A, Table 2).

TABLE 2 Clinical Disease in PLP-EAE in Mice Co-immunized with K6 Day ofDay of Peak Incidence Survival Onset MCS Disease EAE Control 28/28 11/2810.1 ± 0.4 2.9 ± 0.2 13.1 ± 0.5 (n = 28) EAE with 28/28 22/28 12.7 ± 0.51.8 ± 0.2 14.7 ± 0.5 K6- Co-im- munization (n = 28) P = 0.003^(a) P =0.006^(b) P < 0.002^(c) P = 0.036^(b)Clinical Scores in mice immunized with K6 at the time of PLP139-151-EAEinduction. While the incidence of disease did not differ between the twogroups, K6 immunization at the time of PLP139-151 priming was shown todelay the mean day of disease onset, in addition to the day of peakdisease. The mean daily clinical score (MCS) was also significantlyreduced in K6-immunized mice compared with the control group.Additionally, K6-immunization was associated with significantly improvedsurvival to the 21 day end point compared with control mice (^(a)X²;^(b)unpaired Student's t-test; ^(c)Mann-Whitney U test).

The mean daily clinical score after the onset of symptoms was alsosignificantly reduced in K6-immunized mice (1.8±0.3) compared with thecontrol group (2.8±0.4, P<0.01), as was the mean maximal clinicaldisease observed (3.3±0.2 vs. control 4.3±0.2, P=0.003). Over the 21-dayperiod examined, the K6 immunized mice also exhibited significantly moresymptom free days (11.7±0.45 vs. control 9.1±0.38, P≦0.001). SJL micewith high dose PLP139-151-EAE used in these initial experiments,experienced severe clinical disease, with significant mortality.Notably, in this regard, K6-immunization was associated withsignificantly improved survival to the 21-day end point (22/28, 78.6%),compared with (10/28, 35.7%) in the control group (P=0.003, X² Analysis)(FIG. 16B, Table 2). The results of two independent experiments weresimilar and the data were combined for statistical analysis (n=28 pergroup). Given the high incidence of mortality in the high dose PLPexperiments, histological examination of CNS tissue was not performed indetail.

K6 pre-immunization, prior to the time of PLP139-151 priming, similarlyattenuated the mean time to disease onset and the severity of clinicaldisease achieved (FIG. 17, Table 3). K6 pre-immunization delayed themean onset of clinical disease by approximately 4 days relative tocontrol mice (day 14.3±0.8, n=13 vs. PBS-immunized 10.5±0.1, n=15 vs. noprior immunization 10.8±0.2 P<0.001, n=14), as well as the mean time topeak disease (day 16.9±0.6 vs. PBS-immunized 14.7±0.7 vs. no priorimmunization 13.7±0.6 P<0.02). K6-pre-immunization was also associatedwith a significant reduction in the mean daily clinical score after theonset of symptoms (1.4±0.3 vs. PBS-immunized 2.1±0.2 vs. no priorimmunization 2.1±0.2 P<0.001). As was the case for K6 immunization atthe time of EAE induction, K6-pre-immunized mice exhibited significantlymore disease free days over the period examined (13±0.8 vs.PBS-immunized 9.5±0.2 vs. no prior immunization 9.6±0.1 P≦0.001). Nosignificant differences in the timing or severity of clinical diseasewere observed between non-immunized and PBS immunized mice. Theincidence of disease and survival in the lower dose PLP-EAE did notdiffer significantly between the groups examined. Notably, despite thelower dose PLP used in the pre-immunization experiment, and theestablishment of high K6 antibody titers prior to priming, the overallability of the two treatment paradigms to delay the onset of disease andto attenuate its severity, was similar. Collectively, these resultsindicate that K6-immunization is effective in delaying and attenuatingclinical signs of PLP139-151-induced EAE and suggest that other methodsof inhibiting K6 activity would similarly attenuate disease.

TABLE 3 Clinical Disease in PLP-EAE in Mice Pre-immunized with K6 Day ofDay of Peak Incidence Survival Onset MCS Disease EAE Control 14/14 12/1410.8 ± 0.2 2.1 ± 0.2 13.7 ± 0.2 (n = 14) EAE Ribi- 15/15 13/15 10.5 ±0.1 2.1 ± 0.2 14.7 ± 0.2 PBS-Pre-im- munization (n = 15) EAE with 12/1311/13 14.3 ± 0.8 1.2 ± 0.2 16.9 ± 0.2 Ribi-K6-Pre- im- munization (n =15) P < 0.001^(a) P <0.005^(a) P <0.001^(a)Clinical Scores associated with pre-immunization of mice with K6 priorto PLP139-151-EAE induction. K6-pre-immunization prior to PLP-139-151priming reduced the mean clinical score and the day of disease onsetrelative to control mice receiving no prior immunization orpre-immunized with PBS alone (^(a)*Mann Whitney U, P<0.05).

Example 14 K6-Immunization Reduces Clinical and Histological Disease, inAddition to Th1 Immune Responses in Acute PLP139-151 Induced EAE

To further understand the mechanisms by which anti-K6 antibodiesameliorate PLP139-151-induced disease, the effects of K6-immunization onT cell function were examined using in vivo and in vitro approaches,during the acute phase of the disease. The following approaches wereused.

T Cell Proliferation: Spleens were removed at 12 days post-EAE priming,and proliferation assays were carried out in flat-bottomed 96-wellmicroculture plates (Falcon Labware, Oxnared, Calif.), in a total volumeof 200 μl complete Click's medium, using 5×10⁵ spleen cells.Antigen-specific proliferation was assessed in triplicate by theincorporation of ^([3H])TdR (1 μCi/well) during the final 18 hrs of a 96hr culture period, using a PLP139-151 peptide dilutions starting at 10μg/ml. Parallel concentrations of a non-specific antigen, OVA 323-339peptide (ISQAVHAAHAEINEAGR, SEQ ID NO:3) were used as negative controls.As a positive control, Concanavalin A (Sigma) was used at dilutionsstarting at 10 μg/ml. ^([3H])TdR uptake was detected using a Topcountmicroplate scintillation counter (Packard Instrument, Meriden, Conn.).Results were expressed as stimulation index=mean cpm of Ag containingcultures/mean cpm of control cultures without added antigen.

DTH: DTH responses were quantified using a 24 to 48 hr ear-swellingassay, at 9 days post-PLP139-151 priming. Pre-challenge ear thicknesswas determined using a dial gauge micrometer. DTH responses wereelicited by injecting 10 μg of the priming peptide PLP139-151 in 10 μlsaline, into the ventral surface of the right ear and OVA 323-339peptide at the same concentration into the left ear, as outlined in theTMEV section. At 24 and 48 hrs after ear challenge, the increase in earthickness over pre-challenge measurements was determined. Results wereexpressed in units of 10⁻⁴ inches+SEM. Ear swelling responses were theresult of mononuclear cell infiltration and showed typical DTH kinetics(i.e. minimal swelling at 4 hr, maximal swelling at 24-48 hr).

Th1 Cytokine Production: For cytokine analysis, 2.5×10⁶ spleen cellswere cultured in a total volume of 1 ml complete Click's media,supplemented with 10 μg/ml of PLP139-151, or with Click's media alone.Supernatants were harvested at 72 hr and analyzed for IFN-γ and IL-2production by capture ELISA, using capture and detection antibodies, inaddition to cytokine standards, from Pharmingen following themanufacturer's recommendations.

Results: To examine the potential effects of K6-immunization on Th1expansion and/or differentiation, the level of PLP139-151-specificT-cell proliferation and cytokine production were assessed in treatedmice. Recall responses of splenocytes isolated from PLP139-151-primedmice were measured by in vitro proliferation assay at the end of eachexperiment, that is, on day 12 post-priming, upon restimulation invitro. Splenocytes from all PLP139-151 primed mice (FIG. 18A),proliferated in response to PLP139-151 peptide in a dose dependentfashion. The proliferative response to PLP139-151, but not theirrelevant antigen, OVA peptide, was significantly decreased insplenocytes isolated from mice immunized with K6 (n=14), compared withPBS immunized mice (n=14), or mice receiving no prior immunization(n=14), when examined on day 12 after PLP139-151-priming (FIG. 18C).More than 10-fold more PLP-peptide was required to produce equivalentproliferation by splenocytes isolated from K6-immunized mice, comparedto each of the control groups (P≦0.005). These results suggest thatanti-K6 antibodies alter the responsiveness of myelin specific T cellsto disease producing antigen.

PLP-139-151-specific DTH responses in K6-immunized mice: In vivo,PLP139-151-induced ear swelling (DTH), was suppressed in K6-immunizedmice when initiated on Day 9 post-PLP priming, in the mice described inFIG. 18A. The development of DTH to the priming epitope was reduced byapproximately 2.6-fold in K6-immunized, relative to their non-immunized,or adjuvant control immunized littermates, at the 48 hr time point(P≦0.003), (FIG. 18D). A significant DTH response was not observedfollowing the injection of OVA peptide in any of the groups, indicatingthe specificity of the assay. This suggests that anti-K6 antibodies act,at least in part, by inhibiting the effector function of myelin-specificTh1 responses.

K6-immunization blocks the differentiation of PLP139-151-specific Th1cells: To determine the effects of anti-K6 treatment on Th1differentiation, we assessed the ability of PLP-responsive T cells toproduce Th1 cytokines, IFN-γ and IL-2. Secondary in vitro stimulation ofPLP139-151-specific T cells, derived from the spleens of micepre-immunized with K6, or PBS, or from those receiving no priorimmunization, revealed that production of the Th1 cytokine IFN-γ0 wassuppressed by approximately 3-fold in the K6 pre-immunized mice (P<0.05unpaired Student's t-test) relative to control groups (FIG. 19, n=6 pergroup). The levels of IFN-γ production by unstimulated cells, was notsignificantly affected, suggesting recovery of similar numbers ofactivated Th1 cells, and arguing against global immuno-suppression by K6immunization. Moreover, despite reduced levels of IFN-γ, IL-2 secretionwas comparable between the different groups. PLP139-151-inducedsecretion of IL-4, IL5 and IL10 were also assessed, but only minimallevels of secretion of these cytokines were observed in all cultures andthis likely reflects the fact that SJL mice are poor Th2 responders.These results are consistent with the ability of anti-K6 antibodies toinhibit Th1 differentiation.

K6-pre-immunization reduces the development of histological EAE:Detailed histological examination of inflammation and pathologicalchanges in the spinal cord were made in the K6 pre-immunized, andcontrol groups of mice, to determine whether reduced clinical diseasescores seen with K6-immunization, correlated with reduced CNSinflammation and/or pathology. Quantitative evaluation of spinal cordsections showed that reduced clinical deficits were associated withreduced meningeal inflammation and pathology when examined either at day12 (FIG. 18B), or at day 21 (FIG. 17B), following PLP-139-151 priming. Acomparison of the percent of spinal cord quadrants with meningealinflammation, or frank parenchymal pathology, is shown in FIGS. 17B and18B. The mean percent of spinal cord quadrants with meningealinflammation/white matter pathology, at 12 days post-EAE induction, wassignificantly reduced in the K6-immunized mice (37.3%±13.9/15.9%±7.1,n=6), compared to those mice immunized with PBS alone(78.8%±7.3%/40.1±19.9, n=6), and to those receiving no priorimmunization (77.6%±17.5/47.5%±10.4, n=6) (unpaired Student's t-test,P≦0.05). A similar reduction in the percent of spinal cord quadrantswith meningeal inflammation/pathology was also apparent betweenK6-pre-immunized mice on day 21 post-EAE induction (44.6%±6.3/12.5%±3.7,n=11), compared to those mice immunized with PBS alone(77.1%±4.4/36.6%±5.2, n=13), and to those receiving no priorimmunization (76.5%±6.2/47.5%±10.4) (unpaired Student's t-test, P≦0.007,n=12). This was the case, even though clinical deficits were notstatistically different between the different groups of mice at the21-day time point. No significant differences were observed between micepre-immunized with PBS, compared to those receiving no immunization,prior to the induction of EAE, at any of the time points examined.

Example 15 Immunoglobulin from K6-Immunized Mice Blocks the EnzymaticActivity of K6 In Vitro

To determine the efficacy of K6-antibodies generated by directK6-immunization of mice to block K6 activity, immunoglobulins wereisolated from pooled sera of mice immunized with K6 in CFA or with CFAcontaining PBS alone, collected prior to EAE induction, by protein Gpurification (Pharmacia). Immunoglobulin isolated from K6-immunized micewas also compared to commercially available normal mouse IgG (ChromPuremouse IgG, whole molecule, Jackson ImmunoResearch), as an alternativenegative control. Additionally, monoclonal antibodies raised againstwhole recombinant K6 were generated, and their ability to block K6activity in vitro was examined. The following methods were used.

MBP Hydrolysis: The function blocking capacity of K6-antibodies wasexamined in vitro by determining their ability to inhibit cleavage ofrat myelin basic protein (MBP), isolated from whole rat spinal cord, aswell as an Arginine-specific fluorogenic substrate, Ac-Ala-Thr-Arg-pNa(Bachem, King of Prussia, Pa.). For analysis of MBP digestion, 10 ng ofactive-K6 was mixed with 2 μg of IgG isolated from mice immunized withK6 or PBS alone, in 50 mM Tris and 100 mM NaCl buffer (pH 8.0). Thereaction mix was incubated for 15 min at RT. IgG negative controls weresubstituted with an equal volume of suspension buffer alone, in place ofK6. MBP (5 μg) was then added to each reaction tube, or Tris bufferalone, as a control, and the reactions allowed to proceed for 3 hrs at37° C. The reaction was stopped by adding SDS-PAGE sample buffer withβ-mercaptoethanol and analyzed by 20% Tricine SDS-PAGE.

To examine the effect of IgG isolated from the sera of K6-immunized miceto block hydrolysis of the fluorogenic substrate AcATRpNA, 20 ng ofactive K6 was pre-incubated with 4 μg of IgG (1:35 molar ratio) at RTfor 15 min, in 50 mM Tris., 1 mM EDTA, pH 8.5. The kinetic conditions ofthe assay were 1 nM of activated K6 with 400 μM AcATRpNA, incubated at37° C. Absorbances were read at 405 nm at 15 min intervals over a periodof 2 hr, with a final reading at the 3 hr time point, on a Beckmancoulter DU640 spectrophotometer interfaced with temperature controller.A similar experimental design was used to test the ability of twoK6-monoclonal antibodies (K6-2 and K6-3, IgG_(1K)) to block K6-mediatedsubstrate hydrolysis.

Results: The sera from K6 immunized mice were analyzed by ELISA forlevels of K6 antibody production at the conclusion of each experiment,and high levels were determined in each case. The ability of antiserafrom K6-immunized mice to block K6-specific enzymatic activity wasevaluated in vitro (FIG. 20, Table 3). The IgG fraction was isolatedfrom sera of mice immunized with K6, or with adjuvant alone, prior toEAE induction. Pre-incubation of K6 with IgG isolated from K6 immunizedmice effectively blocked K6-mediated hydrolysis of MBP (lane 2), whileIgG isolated from animals immunized with CFA containing only PBS (lane3) did not. Addition of IgG isolated from the different experimentalgroups alone, had no effect on MBP, in the absence of added K6 (lanes 5and 6). The IgG fraction from K6-immunized mice, but not that fromcontrol mice, or that obtained commercially (data not shown), blockedK6-mediated degradation of MBP in vitro (92.3% decrease), (FIG. 20A),and significantly reduced the rate of K6-AcATRpNA substrate hydrolysis(75-87% decrease), (FIG. 20B, Table 4).

TABLE 4 Normalized Reaction Rate against Change Sample (A405/min) K6 (%)(%) K6 alone 0.000804 100 0 K6 + anti-K6 IgG 0.0000619 7.7 −92.3decrease K6 + anti-CFA IgG 0.00106 131.8 31.8 increase anti-K6 IgGcontrol 0.0000458 5.7 −94.3 decrease anti-CFA IgG control 0.0000194 2.4−97.6 decreasePercent change in the rate of K6-mediated AcATRpNA-substrate hydrolysisin the presence of added IgG isolated from K6 immunized mice or frommice immunized with PBS alone (CFA control). There was a 92.3% decreasein substrate hydrolysis with the addition of IgG isolated fromK6-immunized mice, compared to the rate of substrate hydrolysis seen inthe presence of K6 alone. The addition of isolated IgG alone, withoutadded K6, did not result in any significant substrate hydrolysis overtime. The addition of anti-CFA resulted in a slight increase in the rateof substrate hydrolysis over that seen with K6 alone, an effect possiblymediated by the ability of IgG to block the rate of K6 self-autolysis.

Parallel to the ability of IgG isolated from K6-immunized mice to blockthe activity of K6 in vitro, K6-specific monoclonal antibodies blockedK6 mediated substrate hydrolysis (FIG. 21). Pre-incubation of active K6with K6-specific monoclonal antibodies (hK6-2 or hK6-3) for a period 15min at RT (molar ratio of 1:1 or 1:10), each effectively, dramatically,and in a dose dependent fashion, blocked K6-mediated hydrolysis of thefluorogenic AcATRpNA substrate (see Table 5). Readings were taken everymin, over a period of 88 min. K6-specific monoclonal antibodies resultedin greater than a 75% decrease in the rate of substrate hydrolysis. Bycontrast normal mouse IgG (control), did not produce similar decreases,and in fact, resulted in a slight increase in K6-mediated substratehydrolysis by likely decreasing the rate of K6-autolysis.

TABLE 5 Normalized Reaction Rate against Change Sample (A405/min) K6 (%)(%) K6 alone 6.685E−05 100 0 Control IgG + K6 7.675E−05 115 15% (1:1)Control IgG + K6 8.55E−05 128 increase (1:10) K6-2 IgG + K6 (1:1)1.67E−05 25 28 increase K6-2 IgG + K6 (1:10) 8.835E−0 13 −75 decreaseK6-3 IgG + K6 (1:1) 1.58E−05 24 −87 decrease K6-3 IgG + K6 (1:10)8.135E−06 12 −76 decrease −88 decrease

These results indicate that the mechanism of action of directK6-immunization in attenuating disease in the inflammatory animal modelsexamined herein, was likely due to the ability of antibodies generatedby this approach to directly block K6-enzymatic activity, which islikely to play key effector functions in mediating inflammatory disease.Therefore, other methods of inhibiting the functional activity of K6,such as K6-specific protease inhibitors, or antisense strategies, alsocan be used to block inflammatory disease.

Example 16 Immunoglobulins Isolated from K6-Immunized Mice Decrease theMigration of Immune Cells In Vitro

The possibility that K6-specific antibodies generated by directimmunization of mice with K6 block the migration of immune cells, wasexamined by determining the effect of IgG isolated from the sera ofK6-immunized mice on the migration of splenocytes in vitro, using amodified Boyden Chamber assay. Splenocytes were isolated from normalmice and grown in complete Click's media containing 10 μg/mlconcanavilin A (ConA, Sigma). After 48 hr in culture, cells wereharvested and grown for an additional 3 hr in serum starvation media(phenol red free RPMI, 1% bovine serum albumin, 1 mM Hepes Buffer, 50U/ml penicillin-streptoycin and 2 mM glutamine). Cells were labeled withCalcein AM according to the manufacturers recommendations (MolecularProbes). 1×10⁶ Calcein AM labeled cells were applied in 250 μl of serumstarvation media to each of the upper wells of a Matrigel coatedFluoroblock 24 well plate (BD Pharmigen), with or without the additionof 25 μg/ml of IgG from K6 immunized mice, and 750 μl of the same mediawas added to the lower chamber with SDF-1α (50 ng/ml), added as achemoattractant. IgG isolated from CFA immunized mice, or commerciallyavailable purified mouse IgG (Jackson Laboratories), served asimmunoglobulin controls. The fluorescence of cells, which had migratedinto the lower chamber, was read after a 24 hr culture period on aCytoflour 4000 bottom reading plate reader at A530 (BioRad).

Results: A key initiating event in the development of immune-mediatedCNS demyelination, is the extravasation and migration of immune cellsfrom the vascular system into the CNS. As described herein, mice withhigh K6 antibody titers have significantly less spinal cord meningealand parenchymal inflammation in both TMEV and PLP139-151 induced CNSinflammatory disease. In this example, it is demonstrated in vitro thatIgG isolated from K6-immunized mice (FIG. 22), or K6-specific monoclonalantibodies (not shown), inhibit the migration of activated splenocytesin a Boyden Chamber invasion assay. Compared with normal mouse IgG(control) and to the addition of no antibody, the addition of K6-IgGinhibited migration by 25% (*unpaired Student's t-test). These findingsindicate the efficacy of K6-specific antibodies generated in anautonomous fashion, in attenuating clinical disease and spinal cordpathology may, at least in part, be due to a decrease in the ability ofinflammatory cells to migrate into and within the CNS. These resultsadditionally support the idea that other methods of inhibiting theenzymatic activity of K6, would also abrogate inflammatory cellmigration.

Example 17 K6 is Upregulated in Activated Immune Cells

To approach the question of whether K6-production by infiltrating CNSinflammatory cells, represents constitutive expression, or anupregulation in activated cells, the levels of K6 production andsecretion were examined in resting and activated splenocytes, using bothflow cytometry and ELISA techniques.

Splenocytes were isolated from normal SJL mice and grown in the presenceor absence of 10 μg/ml ConA for T cell activation. Cell were harvestedafter 72 hr and examined for intracellular K6-production by flowcytometry, using a K6-specific IgG_(1K) monoclonal antibody (K6-2), andan anti-mouse FITC-labeled secondary. Purified mouse IgG_(1K) was usedas an isotype control. Cells were co-labeled with PE-labeled T cellmarkers, CD4 and CD8 (Pharmingen).

Incubation of cells in the presence of ConA (10 μg/ml) for 72 hr(unfilled areas) resulted in an increase in the number and intensity ofK6 staining, in CD4+ (FIG. 23A) and CD8+ T cells (FIG. 23B), over thatseen in splenocytes cultured in PBS alone.

For examination of K6 production in splenocytes and secretion into thecell culture media, splenocytes isolated from normal SJL mice andcultured in the presence of 10 μg/ml ConA, 5 μg/mL lipolysaccharide(LPS), 20 μg/mL CD3-antibody (CD3-Ab), or PBS alone. Cells and cellculture supernatants, were harvested after a period of 72 hr, andexamined for levels of K6 protein by ELISA. Plates were coated with 100μl of proteins at 250 μg/ml diluted in 0.1 M carbonate buffer, or with200 μl of cell culture supernatant, and incubated overnight at 4° C.After blocking non-specific binding with 1% BSA, K6 was detected withthe K6-specific IgG_(1K) monoclonal antibody (K6-2) and AP-conjugatedanti-mouse IgG (Jackson Laboratories). Plates were read at 405 nm.

As shown in FIG. 24 (left panel), specific activation of T cells (Con Aand CD3 Ab), or non-specific activation of all splenocytes, produced asignificant increase in K6 production, and secretion into the media,compared to those cells grown in PBS alone (FIG. 24 (right panel)). LPSstimulation caused a 2-fold or greater increase in K6 production, whileselective T cell activation resulted in approximately a 1.5-foldincrease in K6. These results indicate that activated immune cells, suchas those seen in CNS-inflammatory lesions, express higher levels of K6than resting immune cells, which we propose contributes to pathogenesis.

Example 18 X-Ray Structure Refinement and Model

A total of 140 solvent molecules were added to the refined hK6structure. One tentatively assigned solvent molecule exhibitedoctahedral coordination geometry with adjacent solvent molecules andshort (˜2.0 Å) contact distances with these groups. This solvent wastherefore assigned as a Mg²⁺ ion 33. Unambiguous density also wasvisible within the active site region, indicating the presence of abound benzamidine inhibitor. In the final refined structure, 227 of the229 amino acid residues were defined in the electron density map. Theobserved electron density is in full agreement with the amino acidsequence deduced from the cDNA sequence. The peptide backbone of hK6could be traced unambiguously from its amino-terminal Ile16 to Gln243(using the chymotrypsinogen numbering scheme). Carboxy-terminal residuesAla244 and Lys245 lacked adequate electron density and were not builtinto the model. The side chain residues of Lys24, Arg110, Gln239, andGln243 were undefined in the electron density map and were thereforemodeled as alanine residues. Asp150 was modeled in multiple rotamerconformations. Some of the loop regions, in particular the region fromTrp215 to Pro225, required extensive rebuilding due to large differencesfrom that of the search model. The model refined to acceptable valuesfor stereochemistry and crystallographic residual (Table 6).

TABLE 6 Crystal, data collection, and refinement statistics A. Crystaldata Space group P212121 Cell dimensions (Å) a = 39.1 b = 62.1 c = 85.8Molecules/asymmetric unit 1 Matthews' constant (Vm) (Å3/Da) 1.80 Maximumresolution (Å) 1.75 B. Data Collection and Processing Total/uniquereflections 495,027/21,777 Completion (43 − 1.75 Å)/(X − 1.75 Å) (%)96.0/82.7 I/σ (43 − 1.75 Å)/(1.79X − 1.75 Å) 43.0/4.9 Rmerge (43 −1.75Å)/ (1.79X − 1.75 Å) (%) 5.7/38.2 Wilson temperature factor (Å2)26.6 C. Refinement Rcrystal (43 − 1.75 Å) (%) 20.9 Rfree (43 − 1.75 Å)(%) 24.1 rms bond length deviation (Å) 0.005 rms bond angle deviation(°) 1.35 rms B-factor deviation (σ) 2.83 Ramachandran plot (%) Mostfavored region 87.6 Additional allowed region 12.4 Generously allowedregion 0 Disallowed region 0 Number of atoms/molecule Non-H protein1,685 Water/ion 139/1

Example 19 Evaluation of Recombinant hK6 Protein

The homogeneity of purified hK6 was evaluated using amino-terminalsequencing and MALDI-TOF mass spectrometry. Mass spectrometry revealedthat the hK6 samples used for crystallization contained intact,glycosylated enzyme. The major peak had a mass of 25,866 Da, which is adifference of 1366 Da from the mass calculated from the proteinsequence. This extra mass corresponds to approximately sixN-acetylglucosamine molecules. Furthermore, peaks corresponding to sixglycosylated forms were visible in the mass spectrum, with the averagedifference in mass between each form being ≈184 Da, which corresponds tothe mass of one hexose unit. Amino-terminal sequencing analysis yieldeda single sequence of Leu-Val-His-Gly, representing the correct aminoterminal sequence for mature hK6.

Example 20 Autolytic Inactivation of hK6

Determination of the x-ray structure of hK6 provides an opportunity tofurther characterize the autolytic regulation of K6/hK6. Unlike themouse kallikreins, and similar to trypsin, autolysis of hK6 leads toinactivation. Thus, autolysis represents a potential regulatorymechanism in controlling the activity of hK6. Arg 76, a site ofautolysis in hK6, is the most solvent accessible arginine residue in thestructure. Both trypsin and hK6 are inactivated by autolysis, andalthough the sites of autolysis are not identical, both proteasesautolyze within the carboxyl terminal domain. The two canonical sites ofautolysis in the mouse kallikreins, which are not associated withinactivation are located within the amino terminal domain (at positions95 and 148). Cleavages within the carboxyl terminal domain may result indestabilization of the structure, and inactivation by autolysis mayrepresent a stability-based mechanism of inactivation. Alternatively,cleavages close to the active site histidine (at position 57) maypromote flexibility at this position that is incompatible with thecatalytic mechanism.

Example 21 Structural Relationship of hK6 with Other Serine Proteases

The secondary structure of hK6 is composed of thirteen β-strands, twoα-helices, two 310-helices, and eight identifiable loop regions. Theseloop regions have varying functions that, based upon the structures ofrelated serine proteases, include defining substrate specificity andautolytic regulation. In addition, these loops can provide sites forN-glycosylation that may serve to regulate activity in this class ofenzymes. When comparing the x-ray structure of hK6 with either bovinetrypsin or a mouse kallikrein (pro-renin converting enzyme, mK13) thereare three immediately identifiable loop regions adjacent to the activesite that exhibit structural heterogeneity. These include residuepositions 92-102 (kallikrein loop), 141-152 and 172-178. The kallikreinloop of hK6 is indistinguishable in length in comparison to thedegradative proteases trypsin and chymotrypsin, and shorter than thatseen in mouse kallikreins or other regulatory type proteases. Althoughthe amino acid sequence within this region differs between hK6 andtrypsin, the structures are essentially identical.

The short surface loop comprising residue positions 172-178 is identicalin length for hK6, rK6, trypsin, chymotrypsin, mK13, neuropsin, ppKa).The amino acid sequence for hK6 within this region is identical to thatof bovine trypsin with the exception of position 178, and adopts anessentially identical structure as bovine trypsin. This short loop isoriented away from the active site, and contrasts with the homologousregion in mK13 (which is oriented towards the active site).

The loop region 141-152 in hK6 is shorter than that in trypsin, andleads to a conformation that orients this loop away from the active sitein comparison to trypsin. In the comparison with other proteases, thebroad-specificity degradative proteases generally have a shorter lengthloop in this region, whereas the regulatory proteases have longer loopsthat afford greater contributions to the substrate binding site.

In general, the structural data for the variable surface loop regionsthat border the active site describe loops that are both short andgenerally oriented away from the substrate binding site. Thus, theircontribution to formation of the S2 and S3 sites within the protease arelimited. This is a characteristic feature of the degradative typeproteases, exemplified by the digestive enzymes trypsin andchymotrypsin.

Kinetic results indicate that hK6 has a 133-fold greater catalyticefficiency for cleavage after arginine compared to cleavage after lysinein a tripeptide substrate. Thus, the specificity of hK6 for an arginineversus lysine at the P1 position is more similar to proteases such asporcine kallikrein, and thrombin which have similar preferences forarginine over lysine at the P1 position, and unlike trypsin which has amuch smaller preference of only 2- to 10-fold. The S1 binding pocket isdefined by residues 189-195, 214-220, and 224-228 and the catalytictriad. Regions 189-195 and 224-228 are identical between hK6 andtrypsin, however, region 214-220 is heterogenous with regard to bothlength and sequence, and is therefore the likely structural determinantof the P1 specificity.

It has been reported that N-linked oligosaccharides within thekallikrein loop of neuropsin affect the size of the S2 pocket and thatmutations in this region result in a significant decrease in both kcatand Km (while maintaining the overall kcat/Km). As previously mentioned,hK6 lacks the equivalent kallikrein loop characteristic of theregulatory proteases, including the N-linked asparagine residue atposition 95. However mass spectrometry data suggests there is apotential N-linked glycosylation site at position Asn132 that is notpresent in any of the other known kallikrein structures. In contrast tothe N-glycosylation site found on the kallikrein loop in otherkallikreins, this site is quite distant from the active site and lies atthe “rear” of the enzyme. There is electron density present in thisregion that is indicative of possible sugar residues. The function ofthis site of glycosylation has yet to be determined, but due to itsdistal location from the active site, it is hypothesized not to affectenzyme specificity or function.

OTHER EMBODIMENTS

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

1. A method for treating multiple sclerosis in a mammal, said methodcomprising administering to said mammal an amount of an antibody havingspecific binding affinity for kallikrein 6 (K6) effective to treat saidmultiple sclerosis, wherein said antibody inhibits the enzyme activityof K6.
 2. The method of claim 1, wherein said method further comprisesmonitoring said multiple sclerosis in said mammal.
 3. The method ofclaim 1, wherein said antibody is polyclonal.
 4. The method of claim 1,wherein said antibody is monoclonal.